Multiplex nucleic acid reactions

ABSTRACT

The invention is directed to a variety of multiplexing methods used to amplify and/or genotype a variety of samples simultaneously.

The present application is a continuation of U.S. application Ser. No.10/194,958, filed on Jul. 12, 2002, which is a continuation-in-part ofU.S. application Ser. No. 10/177,727, filed Jun. 20, 2002, which claimsthe benefit of priority to U.S. Provisional Application Nos. 60/305,118,filed Jul. 12, 2001, 60/311,271, filed Aug. 9, 2001, 60/336,958, filedDec. 3, 2001 and 60/341,827, filed Dec. 17, 2001, and is acontinuation-in-part of each of the following:

-   -   U.S. application Ser. No. 09/779,376, filed Feb. 7, 2001, now        abandoned, which claims the benefit of priority to U.S.        Provisional Application Ser. Nos. 60/180,810, filed Feb. 7, 2000        and 60/234,732, filed Sep. 22, 2000;    -   U.S. application Ser. No. 09/915,231, filed Jul. 24, 2001, now        U.S. Pat. No. 6,890,741, which claims the benefit of priority to        U.S. Provisional Application Ser. Nos. 60/234,143, filed Sep.        21, 2000 and 60/297,609, filed on Jun. 11, 2001 and is a        continuation-in-part of U.S. application Ser. No. 09/779,376,        filed Feb. 7, 2001, now abandoned, which claims the benefit of        priority to U.S. Provisional Application Ser. Nos. 60/180,810,        filed Feb. 7, 2000 and 60/234,732, filed Sep. 22, 2000; and    -   U.S. application Ser. No. 09/931,285, filed Aug. 16, 2001, now        U.S. Pat. No. 6,913,884,        all of which are expressly incorporated herein by reference.

Portions of this invention were made with government support underHG02003 awarded by the National Human Genome Research Institute andCA81952 awarded by the National Cancer Institute. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention is directed to a variety of multiplexing methods used toamplify and/or genotype a variety of samples simultaneously.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acids is an important tool fordiagnostic medicine and molecular biology research. Gene probe assayscurrently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal genes andidentifying mutant genes such as oncogenes, in typing tissue forcompatibility preceding tissue transplantation, in matching tissue orblood samples for forensic medicine, and for exploring homology amonggenes from different species.

Ideally, a gene probe assay should be sensitive, specific and easilyautomatable (for a review, see Nickerson, Current Opinion inBiotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to amplify exponentially a specific nucleic acidsequence before analysis as outlined below (for a review, see Abramsonet al., Current Opinion in Biotechnology, 4:41-47 (1993)).

Currently, a variety of biochips comprising substrates with associatednucleic acids are used in a variety of nucleic acid detection systems,including the detection, quantification, sequence determination andgenotyping of a nucleic acid target sequences. However, samplepreparation for these high density chips remains an issue.

Accordingly, it is an object of the invention to provide a number ofmethods directed to the multiplexing amplification and/or genotypingreactions of target sequences to create amplicons that can subsequentlybe detected on an array.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides a method of detecting target sequences in a sample comprisingproviding a first solid support comprising at least a first and a secondtarget sequence, contacting the first and second target sequences withfirst and second probes, respectively, wherein each of the first andsecond probes comprise a first universal priming site, a target specificdomain substantially complementary to at least a portion of the targetsequence, to form first and second hybridization complexes,respectively, removing unhybridized probes, contacting the first andsecond hybridization complexes with a first enzyme to form modifiedfirst and second probes, respectively contacting the modified first andsecond probes with at least a first primer that hybridizes to theuniversal priming site NTPs, and an extension enzyme, wherein the firstand second modified probes are amplified to form first and secondamplicons, respectively, and detecting the amplicons.

In addition the invention provides a method of detecting targetsequences in a sample comprising providing a first solid supportcomprising at least a first and a second target sequence, contacting thefirst and second target sequences with first and second probes,respectively, wherein each of the first and second probes comprise afirst universal priming site, a target specific domain substantiallycomplementary to at least a portion of the target sequence, to formfirst and second hybridization complexes, respectively, removingunhybridized probes, contacting the first and second probes with atleast a first universal primer that hybridizes to the universal primingsite, NTPs and an extension enzyme, wherein the first and second probesare extended to form first and second modified probes, respectively,contacting the first and second modified probes with at least third andfourth probes, respectively, wherein the modified first and secondprobes comprise a detection position, the third and fourth probes eachcomprise an interrogation position, and a second enzyme, wherein thesecond enzyme only modifies the third and fourth probes if there isperfect complementarity between the bases at the interrogation positionand the detection position, forming third and fourth modified probes,and detecting the third and fourth modified probes.

In addition the invention provides a method comprising providing aplurality of target nucleic acid sequences each comprising from 3′ to 5′a first, second and third target domain, the first target domaincomprising a detection position, the second target domain being at leastone nucleotide contacting the target nucleic acid sequences with sets ofprobes for each target sequence, each set comprising a first probecomprising from 5′ to 3′ a first domain comprising a first universalpriming sequence, and a second domain comprising a sequencesubstantially complementary to the first target domain of a targetsequence, and an interrogation position within the 3′ four terminalbases, a second probe comprising a first domain comprising a sequencesubstantially complementary to the third target domain of a targetsequence, to form a set of first hybridization complexes, contacting thefirst hybridization complexes with an extension enzyme and dNTPs, underconditions whereby if the base at the interrogation positions isperfectly complementary with the bases at the detection positions,extension of the first probes occurs through the second target domainsto form second hybridization complexes, contacting the secondhybridization complexes with a ligase to ligate the extended firstprobes to the second probes to form amplification templates.

In addition the invention provides a multiplex reaction methodcomprising providing a sample comprising at least first and secondtargets hybridizing the first and second targets with first and secondprobes, respectively forming first and second hybridization complexes,respectively, immobilizing the first and second hybridization complexes,washing to remove unhybridized nucleic acids, contacting the first andsecond hybridization complexes with an enzyme, whereby the first andsecond probes are modified forming modified first and second probes,respectively, whereby the modified first and second probes are modifiedto contain first and second interrogation nucleotides that arecomplementary to first and second detection nucleotides in the first andsecond targets, respectively, contacting the modified first and secondprobes with first and second allele specific primers, respectively,whereby the first and second allele specific primers hybridize to themodified first and second probes, respectively, 5′ to the first andsecond interrogation nucleotides, dNTPs, polymerase, whereby the firstand second allele specific primers are modified when a target domain ofthe allele specific primers is perfectly complementary to the modifiedtarget probes to form modified first and second allele specific probes,amplifying the modified first and second allele specific probes to formfirst and second amplicons, and detecting the first and secondamplicons.

In addition the invention provides a method comprising providing aplurality of target nucleic acid sequences each comprising from 3′ to 5′a first, second and third target domain, the first target domaincomprising a detection position, the second target domain being at leastone nucleotide, contacting the target nucleic acid sequences with setsof probes for each target sequence, each set comprising:

a first probe comprising from 5′ to 3′, a first domain comprising afirst universal priming sequence, and a second domain comprising asequence substantially complementary to the first target domain of atarget sequence, and an interrogation position within the 3′ fourterminal bases, a second probe comprising a first domain comprising asequence substantially complementary to the third target domain of atarget sequence, to form a set of first hybridization complexes,contacting the first hybridization complexes with at least a firstuniversal primer that hybridize to the first universal priming sequence,an extension enzyme and dNTPs, under conditions whereby if the base atthe interrogation positions are perfectly complementary with the basesat the detection positions, extension of the first probes occurs throughthe second target domains to form second hybridization complexes,contacting the second hybridization complexes with a ligase to ligatethe extended first probes to the second probes to form amplificationtemplates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict a schematic of a preferred embodiment of theinvention. The primary steps of the method include annealingoligonucleotides to immobilized target (e.g. genomic) DNA, a chainextension reaction that is terminated by tagged (e.g. biotinylated)ddNTPs, isolation and amplification of the tagged extension products.

FIG. 2 depicts a preferred method of obtaining templates for single baseextension reaction analysis. The four major phases are 1) FirstExtension from target (gDNA), 2) Second Extension, 3) PCR Amplification,and 4) Allele Specific 4-Dye Single Base Extension.

FIG. 3 depicts a preferred method for complexity reduction and alleleselectivity. The locus specific primer hybridizes upstream of theinterrogation site. It does not have to be directly adjacent to theinterrogation site. The locus specific primer also contains an adaptersequence and universal PCR primer hybridization site. The allelespecific primers are designed to the opposite strand of DNA (seediagram) and the 3′ ends of the primers correspond to the alleles thatare interrogated. The 5′ ends of the allele specific primers arehybridization sites for universal PCR primers.

Tagged locus specific primers are annealed to the genomic DNA andwashed. DNA polymerase (Taq DNA polymerase), dNTPs, ddNTPs and buffer isadded to the hybridized primers. The DNA polymerase will extend thelocus specific primers that have hybridized and are matched exactly atthe 3′ end to DNA. In this first primer extension reaction, the primerextended product has captured the locus allele information and alsoadjacent DNA sequence information. The primer extension products areeluted away from the genomic DNA. The eluted primer extension productsare captured onto another set of streptavidin coated beads through thebiotin molecule on the locus specific primer. This capture processpurifies the primer extension product and reduces the complexity of DNAgoing into the second hybridization and extension process. The secondcapture process may improve the multiplexability of this assay throughthe reduction of complexity.

Allele specific primers for each interrogated locus are added to thecaptured DNA and a second hybridization and wash is performed (at highstringency). DNA polymerase (Taq DNA polymerase), dNTPs, and buffer areadded to the hybridized primers. An extension reaction is carried out.The extended products are eluted and used in a PCR amplificationreaction (using the universal PCR primers specific for these oligos U1,U2 and U3). U2 and U3 are labeled with different fluorescent tags. Theratio in the amount of one allele relative to another is determined bythe ratio of the fluorescent tags.

FIG. 4 depicts an alternative embodiment of the method outlined in FIG.3. An allele specific hybridization approach for allele determinationmay be used in conjunction with the first hybridization, wash andextension. In this process, the locus specific primer is hybridized,washed and extended as above. The locus specific primer does not containadapter sequences or universal primer sequences. The allele specificoligonucleotide contains the universal PCR primer sequences. Allelespecific oligonucleotides are added to the extended products, hybridizedand washed under stringent conditions. Allele specifically hybridizedsequences are retained and later eluted for a PCR reaction.

FIG. 5 depicts an alternative embodiment of the method outlined in FIG.3. In this embodiment allele specific extension is followed by locusspecific extension.

FIG. 6 depicts an alternative embodiment of the method outlined in FIG.3. A second level of allele specificity along with locus specificity maybe obtained by using allele specific extension primers in the secondextension step of FIG. 5. Using allele specific extension primers (onalternate strands) in both extension steps would protect against any 3′to 5′ exonuclease activity acting in the first allele specific extensionstep. The extension products from this approach would be placed into twoseparate PCR reactions containing universal PCR primers specific foreach allele set. Misextensions due to exonuclease activity in the firstor second extension steps would not be amplified.

FIG. 7 depicts a preferred method of solid-phase allele-specific primerextension genotyping. For each locus, two allele specificoligonucleotides are designed with each allele represented by a uniqueadapter. The 3′ end of the allele specific oligonucleotides extend oneor more bases beyond the query site. The oligonucleotides are hybridizedto the template on solid phase under stringent conditions. The solidphase is washed to remove improperly hybridized oligonucleotides. Theresulting complex is then extended by a polymerase in an allele specificmanner. That is a mismatch at the query site will prevent efficientextension.

FIG. 8 depicts an alternative method of labeling as compared to FIG. 7.

FIG. 9 depicts a schematic of universal allele specificoligonucleotides.

FIG. 10 depicts a method using the universal allele specificoligonucleotides described in FIG. 9. In this case, since extension mustoccur from 5′ to 3′, the U4 and U5 sequences are shown at the 3′ end ofthe template, associated with the allele-selective bases.

FIG. 11 depicts a method of removing non-hybridized nucleic acids bynuclease treatment. That is, the complexity of a nucleic acid sample isinitially reduced by hybridization capture with gene specificoligonucleotides. Excess nucleic acid sequences are removed by a singlestranded nuclease.

FIGS. 12A and 12B depict the ICAN amplification scheme.

FIGS. 13 12A and 13B depict a preferred multiplex scheme. Two primershybridize to a target nucleic acid. The primers include target specificportions and universal priming sites. In addition, one of the primers,preferably the upstream primer, includes an allele specific sequence andan adapter sequence that is specific for the particular allele specificsequence. The primers do not hybridize contiguously on the target.Following hybridization the primer is extended with dNTPs and apolymerase. Following primer extension, the upstream and downstreamprimers are ligated. The ligated product is them amplified withuniversal primers that hybridize to the universal priming sites on theprimers resulting in the formation amplicons. Amplicons are labeled witheither labeled primers or labeled dNTPs and detected as an indication ofthe presence of a particular allele.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a variety of compositions andmethods directed to multiplexed analysis of nucleic acids. In apreferred embodiment the methods are directed to multiplexing of nucleicacid detection, genotyping and amplification reactions. While a largebody of literature and methods exist for the use of high densitybiochips comprising nucleic acids, the preparation of samples containingtarget sequences to place on the biochips has not been significantlymultiplexed to allow true high throughput methodologies. The presentinvention is directed to the use of a variety of methods that allow themultiplexed amplification of target sequences prior to detection by anyof a variety of methods including placement on an array for detection,mass spectrometry, electrophoretic techniques, FACS analysis, and thelike.

In general, the method includes a complexity reduction component, aspecificity step and an amplification step. Preferably complexityreduction is performed first. This is followed, in some embodiments, bythe genotyping reaction, followed by multiplexed amplification.Generally, the specificity step includes an enzymatic reaction such as agenotyping reaction as described below. Alternatively, the multiplexedamplification reaction is done first, i.e. following complexityreduction, followed by a genotyping reaction. In both instances, theresulting amplicons are then detected, by a variety of detection methodsincluding utilizing solid support arrays (both random and ordered),liquid arrays, or using technologies such as FACS sorting or massspectroscopy.

Accordingly, the present invention relates to the multiplexamplification and detection of target analytes in a sample. As usedherein, the phrase “multiplex” or grammatical equivalents refers to thedetection, analysis or amplification of more than one target sequence ofinterest. In one embodiment multiplex refers to at least 100 or 200different target sequences while at least 500 different target sequencesis preferred. More preferred is at least 1000, with more than 5000 or10,000 particularly preferred and more than 50,000 or 100,000 mostpreferred. Detection is performed on a variety of platforms as describedherein.

Accordingly, the present invention provides methods for the detection ofnucleic acid target sequences in a sample. As will be appreciated bythose in the art, the sample solution may comprise any number of things,including, but not limited to, bodily fluids (including, but not limitedto, blood, urine, serum, lymph, saliva, anal and vaginal secretions,perspiration and semen, of virtually any organism, with mammaliansamples being preferred and human samples being particularly preferred);environmental samples (including, but not limited to, air, agricultural,water and soil samples); biological warfare agent samples; researchsamples; purified samples, such as purified genomic DNA, RNA, proteins,etc.; raw samples (bacteria, virus, genomic DNA, etc.). As will beappreciated by those in the art, virtually any experimental manipulationmay have been done on the sample.

If required, the target sequence is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, sonication, electroporation, etc., with purification andamplification as outlined below occurring as needed, as will beappreciated by those in the art. In addition, the reactions outlinedherein may be accomplished in a variety of ways, as will be appreciatedby those in the art. Components of the reaction may be addedsimultaneously, or sequentially, in any order, with preferredembodiments outlined below. In addition, the reaction may include avariety of other reagents which may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc., which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

In addition, when nucleic acids are to be detected preferred methodsutilize cutting or shearing techniques to cut the nucleic acid samplecontaining the target sequence into a size that will facilitate handlingand hybridization to the target, particularly for genomic DNA samples.This may be accomplished by shearing the nucleic acid through mechanicalforces (e.g. sonication) or by cleaving the nucleic acid usingrestriction endonucleases, or any other methods known in the art.

In addition, in most embodiments, double stranded target nucleic acidsare denatured to render them single stranded so as to permithybridization of the primers and other probes of the invention. Apreferred embodiment utilizes a thermal step, generally by raising thetemperature of the reaction to about 95° C., although pH changes andother techniques may also be used.

The present invention provides compositions and methods for detectingthe presence or absence of target nucleic acid sequences in a sample. By“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10): 1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of labels, or to increase the stability and half-life ofsuch molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thusfor example the individual units of a peptide nucleic acid, eachcontaining a base, are referred to herein as a nucleoside.

The compositions and methods of the invention are directed to themulti-plexed detection of target sequences. The term “target sequence”or “target nucleic acid” or grammatical equivalents herein means anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA and rRNA, or others. A preferred embodimentutilizes genomic DNA as the primary target sequence.

As is outlined herein, the target sequence may be a target sequence froma sample, or a secondary target such as a product of a reaction such asa detection sequence from an invasive cleavage reaction, a ligated probefrom an OLA reaction, an extended probe from a PCR reaction, or PCRamplification product, (“amplicon”) etc.

The target sequence may be any length, with the understanding thatlonger sequences are more specific. As will be appreciated by those inthe art, the complementary target sequence may take many forms. Forexample, it may be contained within a larger nucleic acid sequence, i.e.all or part of a gene or mRNA, a restriction fragment of a plasmid orgenomic DNA, among others. As is outlined more fully below, probes aremade to hybridize to target sequences to determine the presence orabsence of the target sequence in a sample. Generally speaking, thisterm will be understood by those skilled in the art. The target sequencemay also be comprised of different target domains; for example, in“sandwich” type assays as outlined below, a first target domain of thesample target sequence may hybridize to a capture probe or a portion ofcapture extender probe, a second target domain may hybridize to aportion of an amplifier probe, a label probe, or a different capture orcapture extender probe, etc. In addition, the target domains may beadjacent (i.e. contiguous) or separated. For example, when LCRtechniques are used, a first primer may hybridize to a first targetdomain and a second primer may hybridize to a second target domain;either the domains are adjacent, or they may be separated by one or morenucleotides, coupled with the use of a polymerase and dNTPs, as is morefully outlined below.

The terms “first” and “second” are not meant to confer an orientation ofthe sequences with respect to the 5′-3′ orientation of the targetsequence. For example, assuming a 5′-3′ orientation of the complementarytarget sequence, the first target domain may be located either 5′ to thesecond domain, or 3′ to the second domain.

As outlined herein, in preferred embodiments the target sequencecomprises a position for which sequence information is desired,generally referred to herein as the “detection position” or “detectionlocus”. In a preferred embodiment, the detection position is a singlenucleotide, although in some embodiments, it may comprise a plurality ofnucleotides, either contiguous with each other or separated by one ormore nucleotides. By “plurality” as used herein is meant at least two.As used herein, the base which basepairs with a detection position basein a hybrid is termed a “readout position” or an “interrogationposition”; thus many of the first or second step probes of the inventioncomprise an interrogation position.

In some embodiments, as is outlined herein, the target sequence may notbe the sample target sequence but instead is a product of a reactionherein, sometimes referred to herein as a “secondary” or “derivative”target sequence, or an “amplicon”.

Accordingly, in a preferred embodiment the present multiplexed detectionscheme includes at least one complexity reduction component, at leastone specificity component and at least one amplification component. Inaddition, the method includes detection of the product of the reaction.

The methods of the invention can take on a wide variety ofconfigurations, as are shown in the figures and described in more detailbelow. Generally these components include a complexity reductioncomponent, a specificity component and an amplification component. Thecomponents can be configured in a variety of ways as disclosed below.That is, in one embodiment a complexity reduction step is firstperformed. This is followed by either the amplification or specificitystep. Alternatively, the specificity step is performed first. This canbe followed by the complexity reduction or amplification step.Alternatively, amplification is first performed. This is followed by thecomplexity and specificity steps.

While the above indicates that each of the three components can beperformed in any order. One of skill in the art will appreciate thatwhen amplification is performed first, there will likely be some degreeof complexity reduction or specificity involved. In addition, whenspecificity components are performed first, there will be a degree ofcomplexity reduction. In addition, in some embodiments whenamplification is first performed, there will be some degree ofspecificity and complexity reduction. However, as described below, themethod generally includes three components.

Probes and Primers

As one of skill in the art appreciates, there are several probes orprimers that are used in the present invention. These probes/primers cantake on a variety of configurations and may have a variety of structuralcomponents described in more detail below. The first step probe may beeither an allele specific probe or locus specific probe. By “allelespecific” probe or primer is meant a probe or primer that eitherhybridizes to a target sequence and discriminates between alleles orhybridizes to a target sequence and is modified in an allele specificmanner. By “locus specific” probe or primer is meant a probe or primerthat hybridizes to a target sequence in a locus specific manner, butdoes not necessarily discriminate between alleles. A locus specificprimer also may be modified, i.e. extended as described below, such thatit includes information about a particular allele, but the locusspecific primer does not discriminate between alleles.

In many embodiments, the probes or primers comprise one or moreuniversal priming site(s) and/or adapters, both of which are describedbelow.

The size of the primer and probe nucleic acid may vary, as will beappreciated by those in the art with each portion of the probe and thetotal length of the probe in general varying from 5 to 500 nucleotidesin length. Each portion is preferably between 10 and 100 beingpreferred, between 15 and 50 being particularly preferred, and from 10to 35 being especially preferred, depending on the use and amplificationtechnique. Thus, for example, the universal priming site(s) of theprobes are each preferably about 15-20 nucleotides in length, with 18being especially preferred. The adapter sequences of the probes arepreferably from 15-25 nucleotides in length, with 20 being especiallypreferred. The target specific portion of the probe is preferably from15-50 nucleotides in length. In addition, the primer may include anadditional amplification priming site. In a preferred embodiment theadditional amplification priming site is a T7 RNA polymerase primingsite.

In a preferred embodiment, the allele or locus specific probe or probescomprises a target domain substantially complementary to a first domainof the target sequence. In general, probes of the present invention aredesigned to be complementary to a target sequence (either the targetsequence of the sample or to other probe sequences, as is describedherein), such that hybridization of the target and the probes of thepresent invention occurs. This complementarity need not be perfect;there may be any number of base pair mismatches that will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under the selected reaction conditions.

In one embodiment the target specific portion includes a combinatorialmixture of each nucleotide at each position. In addition the primerincludes a universal priming sequence and an allele specific position.Preferably the universal priming sequence is specific for the particularnucleotide at the allele specific position. That is, in this embodimentthe locus-specific allele selectivity portions of the primer arereplaced with a universal targeting domain that includes region whereeach position is represented by a combinatorial mixture of nucleotides.One of the positions in the universal region (not necessarily the 3′position) is paired with the allele or SNP to be analyzed. The base atthis position is associated with an identifier such as a particularadapter in the primer or with a particular universal priming sequence inthe primer (FIG. 9).

In a preferred configuration, each of the four bases is associated witha different sequence, i.e. universal priming sequence, each sequencehaving similar amplification efficiencies. For amplification, each ofthe four primers is labeled with a different label. In an alternateembodiment it is possible to substitute a universal, i.e. promiscuous(inosine, for example) base at one or more positions in the universalsequence. The primer finds use in extension reactions and ligationreactions as described herein. In addition the primers find use inlinear amplification schemes as depicted in FIG. 10. It should be notedthat one advantage of using the universal targeting domain is thatshorter oligonucleotides can be used. Thus, when universal targetdomains are used, these domains are preferably from about 5 to 15nucleotides in length with from 7 to 10 being particularly preferred.

In a preferred embodiment, one of the probes further comprises anadapter sequence, (sometimes referred to in the art as “zip codes” or“bar codes”). Adapters facilitate immobilization of probes to allow theuse of “universal arrays”. That is, arrays (either solid phase or liquidphase arrays) are generated that contain capture probes that are nottarget specific, but rather specific to individual (preferably)artificial adapter sequences.

Thus, an “adapter sequence” is a nucleic acid that is generally notnative to the target sequence, i.e. is exogenous, but is added orattached to the target sequence. It should be noted that in thiscontext, the “target sequence” can include the primary sample targetsequence, or can be a derivative target such as a reactant or product ofthe reactions outlined herein; thus for example, the target sequence canbe a PCR product, a first ligation probe or a ligated probe in an OLAreaction, etc. The terms “barcodes”, “adapters”, “addresses”, tags” and“zipcodes” have all been used to describe artificial sequences that areadded to amplicons to allow separation of nucleic acid fragment pools.One preferred form of adapters are hybridization adapters. In thisembodiment adapters are chosen so as to allow hybridization to thecomplementary capture probes on a surface of an array. Adapters serve asunique identifiers of the probe and thus of the target sequence. Ingeneral, sets of adapters and the corresponding capture probes on arraysare developed to minimize cross-hybridization with both each other andother components of the reaction mixtures, including the targetsequences and sequences on the larger nucleic acid sequences outside ofthe target sequences (e.g. to sequences within genomic DNA). Other formsof adapters are mass tags that can be separated using mass spectroscopy,electrophoretic tags that can be separated based on electrophoreticmobility, etc. Some adapter sequences are outlined in U.S. Ser. No.09/940,185, filed Aug. 27, 2001, hereby incorporated by reference in itsentirety. Preferred adapters are those that meet the following criteria.They are not found in a genome, preferably a human genome, and they donot have undesirable structures, such as hairpin loops.

As will be appreciated by those in the art, the attachment, or joining,of the adapter sequence to the target sequence can be done in a varietyof ways. In a preferred embodiment, the adapter sequences are added tothe primers of the reaction (extension primers, amplification primers,readout probes, genotyping primers, Rolling Circle primers, etc.) duringthe chemical synthesis of the primers. The adapter then gets added tothe reaction product during the reaction; for example, the primer getsextended using a polymerase to form the new target sequence that nowcontains an adapter sequence. Alternatively, the adapter sequences canbe added enzymatically. Furthermore, the adapter can be attached to thetarget after synthesis; this post-synthesis attachment can be eithercovalent or non-covalent. In a preferred embodiment the adapter is addedto the target sequence or associated with a particular allele during anenzymatic step. That is, to achieve the level of specificity necessaryfor highly multiplexed reactions, the product of the specificity orallele specific reaction preferably also includes at least one adaptersequence.

In this embodiment, one or more of the specificity primers comprises afirst portion comprising the adapter sequence and a second portioncomprising the priming sequence. Extending the amplification primer asis well known in the art results in target sequences that comprise theadapter sequences. The adapter sequences are designed to besubstantially complementary to capture probes.

In addition, as will be appreciated by those in the art, the adapter canbe attached either on the 3′ or 5′ ends, or in an internal position,depending on the configuration of the system, as generally outlined inthe figures.

In one embodiment the use of adapter sequences allow the creation ofmore “universal” surfaces; that is, one standard array, comprising afinite set of capture probes can be made and used in any application.The end-user can customize the array by designing different solubletarget probes, which, as will be appreciated by those in the art, isgenerally simpler and less costly. In a preferred embodiment, an arrayof different and usually artificial capture probes are made; that is,the capture probes do not have complementarity to known targetsequences. The adapter sequences can then be incorporated in the targetprobes.

As will be appreciated by those in the art, the length of the adaptersequences will vary, depending on the desired “strength” of binding andthe number of different adapters desired. In a preferred embodiment,adapter sequences range from about 6 to about 500 basepairs in length,with from about 8 to about 100 being preferred, and from about 10 toabout 25 being particularly preferred.

In a preferred embodiment, the adapter sequence uniquely identifies thetarget analyte to which the target probe binds. That is, while theadapter sequence need not bind itself to the target analyte, the systemallows for identification of the target analyte by detecting thepresence of the adapter. Accordingly, following a binding orhybridization assay and washing, the probes including the adapters areamplified. Detection of the adapter then serves as an indication of thepresence of the target analyte.

In one embodiment the adapter includes both an identifier region and aregion that is complementary to capture probes on a universal array asdescribed above. In this embodiment, the amplicon hybridizes to captureprobes on a universal array. Detection of the adapter is accomplishedfollowing hybridization with a probe that is complementary to theadapter sequence. Preferably the probe is labeled as described herein.

In general, unique adapter sequences are used for each unique targetanalyte. That is, the elucidation or detection of a particular adaptersequence allows the identification of the target analyte to which thetarget probe containing that adapter sequence bound. However, in somecases, it is possible to “reuse” adapter sequences and have more thanone target analyte share an adapter sequence.

In a preferred embodiment the adapters contain different sequences orproperties that are indicative of a particular target molecule. That is,each adapter uniquely identifies a target sequence. As described above,the adapters are amplified to form amplicons. The adapter is detected asan indication of the presence of the target analyte, i.e. the particulartarget nucleic acid.

The use of adapters in combination with amplification following aspecific binding event allows for highly multiplexed reactions to beperformed.

Also, the probes are constructed so as to contain the necessary primingsite or sites for the subsequent amplification scheme. In a preferredembodiment the priming sites are universal priming sites. By “universalpriming site” or “universal priming sequences” herein is meant asequence of the probe that will bind a primer for amplification.

In a preferred embodiment, one universal priming sequence or site isused. In this embodiment, a preferred universal priming sequence is theRNA polymerase T7 sequence, that allows the T7 RNA polymerase make RNAcopies of the adapter sequence as outlined below. Additional disclosureregarding the use of T7 RNA polymerase is found in U.S. Pat. Nos.6,291,170, 5,891,636, 5,716,785, 5,545,522, 5,922,553, 6,225,060 and5,514,545, all of which are expressly incorporated herein by reference.

In a preferred embodiment, for example when amplification methodsrequiring two primers such as PCR are used, each probe preferablycomprises an upstream universal priming site (UUP) and a downstreamuniversal priming site (DUP). Again, “upstream” and “downstream” are notmeant to convey a particular 5′-3′ orientation, and will depend on theorientation of the system. Preferably, only a single UUP sequence and asingle DUP sequence is used in a probe set, although as will beappreciated by those in the art, different assays or differentmultiplexing analysis may utilize a plurality of universal primingsequences. In some embodiments probe sets may comprise differentuniversal priming sequences. In addition, the universal priming sitesare preferably located at the 5′ and 3′ termini of the target probe (orthe ligated probe), as only sequences flanked by priming sequences willbe amplified.

In addition, universal priming sequences are generally chosen to be asunique as possible given the particular assays and host genomes toensure specificity of the assay. However, as will be appreciated bythose in the art, sets of priming sequences/primers may be used; thatis, one reaction may utilize 500 target probes with a first primingsequence or set of sequences, and an additional 500 probes with a secondsequence or set of sequences.

As will be appreciated by those in the art, when two priming sequencesare used, the orientation of the two priming sites is generallydifferent. That is, one PCR primer will directly hybridize to the firstpriming site, while the other PCR primer will hybridize to thecomplement of the second priming site. Stated differently, the firstpriming site is in sense orientation, and the second priming site is inantisense orientation.

As will be appreciated by those in the art, in general, highlymultiplexed reactions can be performed, with all of the universalpriming sites being the same for all reactions. Alternatively, “sets” ofuniversal priming sites and corresponding probes can be used, eithersimultaneously or sequentially. The universal priming sites are used toamplify the modified probes to form a plurality of amplicons that arethen detected in a variety of ways, as outlined herein. In preferredembodiments, one of the universal priming sites is a T7 site. In someembodiments this priming site serves as a template for the synthesis ofRNA.

Accordingly, the present invention provides first target probe sets. By“probe set” herein is meant a plurality of target probes that are usedin a particular multiplexed assay. In this context, plurality means atleast two, with more than 10 being preferred, depending on the assay,sample and purpose of the test. In one embodiment the probe set includesmore than 100, with more than 500 probes being preferred and more than1000 being particularly preferred. In a particularly preferredembodiment each probe contains at least 5000, with more than 10,000probes being most preferred.

Accordingly, the present invention provides first target probe sets thateach comprise at least a first universal priming site.

In a preferred embodiment, the target probe may also comprise a labelsequence, i.e. a sequence that can be used to bind label probes and issubstantially complementary to a label probe. This system is sometimesreferred to in the art as “sandwich-type” assays. That is, byincorporating a label sequence into the target probe, which is thenamplified and present in the amplicons, a label probe comprising primary(or secondary) detection labels can be added to the mixture, eitherbefore addition to the array or after. This allows the use of highconcentrations of label probes for efficient hybridization. In oneembodiment, it is possible to use the same label sequence and labelprobe for all target probes on an array; alternatively, different targetprobes can have a different label sequence. Similarly, the use ofdifferent label sequences can facilitate quality control; for example,one label sequence (and one color) can be used for one strand of thetarget, and a different label sequence (with a different color) for theother; only if both colors are present at the same basic level is apositive called.

Thus, the present invention provides target probes that comprise any,all or any combination of universal priming sequences, bioactive agents(e.g. target specific portion(s)), adapter sequence(s), optionally anadditional amplification priming sequence such as T7 RNA primingsequence and optionally label sequences. These target probes are thenadded to the target sequences to form hybridization complexes. As willbe appreciated by those in the art, when nucleic acids are the target,the hybridization complexes contain portions that are double stranded(the target-specific sequences of the target probes hybridized to aportion of the target sequence) and portions that are single stranded(the ends of the target probes comprising the universal primingsequences and the adapter sequences, and any unhybridized portion of thetarget sequence, such as poly(A) tails, as outlined herein).

Complexity Reduction

Complexity reduction is a principal component of the multiplex schemeset forth herein. Generally, complexity reduction is a method forenriching for a particular target or locus. That is, complexityreduction is considered a method that results in removal of non-targetnucleic acids from the sample or removal of probes/primers that have nothybridized correctly or at all to a target nucleic acid. In addition,complexity reduction includes removal of probes that have not beenmodified during a enzymatic step. That is, complexity reduction includesremoving non-target nucleic acids, i.e. enriching for target nucleicacids or removing non-hybridized probes or primers prior to an enzymaticstep, i.e. either an amplification or specificity step, or both.

There are a variety of ways one can include a complexity reduction step.These include, but are not limited to, selective immobilization oftarget nucleic acids or probes/primers that are modified in a targetspecific manner, selective removal of non-target nucleic acids, andselective destruction of non-target nucleic acids. Such destructionincludes but is not limited to denaturation, degradation or cleavage ofnon-target nucleic acids. In addition, complexity reduction can includecomponents such as target selective amplification, although this alsoincludes amplification and components.

In a preferred embodiment complexity reduction is accomplished byselectively immobilizing a primer that has been modified in a targetspecific manner. That is, either locus specific or allele specificprimers are hybridized with a target. The target can be immobilized orin solution. Following hybridization, the primer is extended in a primerextension reaction. Preferably either the primer or NTPs include apurification tag as described herein that allows for removal orpurification of the extended product from the reaction mixture. Onceextended, generally the modified primer is immobilized on a solidsupport as described herein. Following immobilization of the modifiedprimer, the support is washed to remove both non-target nucleic acidsand primers that were not modified, i.e. extended. The immobilizedprimers, thus, include information about the target locus includingparticular allelic information. This results in enrichment of targetnucleic acids or removal of non-target nucleic acids.

In a preferred embodiment the complexity reduction component includesselective immobilization of target nucleic acids. That is, targetnucleic acids are preferentially immobilized on a solid support ratherthan non-target nucleic acids.

In this embodiment target DNA is preferably reduced in size initially.This is easily accomplished by methods as known in the art such as, butnot limited to, shearing or cleaving with restriction enzymes. Thetarget nucleic acid is contacted with probes that hybridize to thetargets. Preferably the hybridization is performed under low stringencyconditions such that the probes do not discriminate between alleles of aparticular locus. The resulting complexes are then immobilized on asupport. In a preferred embodiment the probes are labeled with apurification tag as described herein to allow for immobilization.Following immobilization, the support is washed to remove non-hybridizedtargets, while leaving targets that are substantially complementary tothe probes immobilized on the solid support. After removal ofnon-hybridized probes, the target nucleic acids can be removed with astringent wash. This allows for enrichment of target sequences that arethen available for further analysis.

In one embodiment, the target sequence, probe or primer, includingmodified primer, is attached to a first solid support. By “substrate” or“solid support” or other grammatical equivalents herein is meant anymaterial that is appropriate for or can be modified to be appropriatefor the attachment of the target sequences. As will be appreciated bythose in the art, the number of possible substrates is very large.Possible substrates include, but are not limited to, glass and modifiedor functionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon ornitrocellulose, ceramics, resins, silica or silica-based materialsincluding silicon and modified silicon, carbon, metals, inorganicglasses, plastics, optical fiber bundles, and a variety of otherpolymers. Magnetic beads and high throughput microtier plates areparticularly preferred.

The composition and geometry of the solid support vary with its use. Inthis particular embodiment, supports comprising microspheres or beadsare preferred for the first solid support. By “microspheres” or “beads”or “particles” or grammatical equivalents herein is meant small discreteparticles. The composition of the beads will vary, depending on theclass of bioactive agent and the method of synthesis. Suitable beadcompositions include those used in peptide, nucleic acid and organicmoiety synthesis, including, but not limited to, plastics, ceramics,glass, polystyrene, methylstyrene, acrylic polymers, paramagneticmaterials, thoria sol, carbon graphited, titanium dioxide, latex orcross-linked dextrans such as Sepharose, cellulose, nylon, cross-linkedmicelles and teflon, as well as any other materials outlined herein forsolid supports may all be used. “Microsphere Detection Guide” from BangsLaboratories, Fishers Ind. is a helpful guide. Preferably, in thisembodiment, when complexity reduction is performed, the microspheres aremagnetic microspheres or beads.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for assay. The bead sizes range from nanometers, i.e.100 nm, to millimeters, i.e. 1 mm, with beads from about 0.2 micron toabout 200 microns being preferred, and from about 0.5 to about 5 micronbeing particularly preferred, although in some embodiments smaller beadsmay be used.

The target sequence, probe or primer is attached to the first solidsupport in a number of ways. In a preferred embodiment, purificationtags are used. By “purification tag” herein is meant a moiety which canbe used to purify a strand of nucleic acid, usually via attachment to asolid support as outlined herein. Suitable purification tags includemembers of binding partner pairs. For example, the tag may be a haptenor antigen, which will bind its binding partner. In a preferredembodiment, the binding partner can be attached to a solid support asdepicted herein and in the figures. For example, suitable bindingpartner pairs include, but are not limited to: antigens (such asproteins (including peptides)) and antibodies (including fragmentsthereof (FAbs, etc.)); proteins and small molecules, includingbiotin/streptavidin; enzymes and substrates or inhibitors; otherprotein-protein interacting pairs; receptor-ligands; and carbohydratesand their binding partners. Nucleic acid—nucleic acid binding proteinspairs are also useful. In general, the smaller of the pair is attachedto the NTP for incorporation into the primer. Preferred binding partnerpairs include, but are not limited to, biotin (or imino-biotin) andstreptavidin, digeoxinin and Abs, and Prolinx™ reagents (seewww.prolinxinc.com/ie4/home.hmtl).

In a preferred embodiment, the binding partner pair comprises biotin orimino-biotin and streptavidin. Imino-biotin is particularly preferred asimino-biotin disassociates from streptavidin in pH 4.0 buffer whilebiotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or90% formamide at 95° C.).

Additional techniques include, but are not limited to, enzymaticattachment, chemical attachment, photochemistry or thermal attachmentand absorption.

In a preferred embodiment, as outlined herein, enzymatic techniques areused to attach the target nucleic acid, probe or primer to the support.For example, terminal transferase end-labeling techniques can be used asoutlined above; see Hermanson, Bioconjugate Techniques, San Diego,Academic Press, pp 640-643. In this embodiment, a nucleotide labeledwith a secondary label (e.g. a binding ligand, such as biotin) is addedto a terminus of the target nucleic acid; supports coated or containingthe binding partner (e.g. streptavidin) can thus be used to immobilizethe target nucleic acid. Alternatively, the terminal transferase can beused to add nucleotides with special chemical functionalities that canbe specifically coupled to a support. Preferred embodiments utilize theaddition of biotinylated nucleotides followed by capture on streptavidincoated magnetic beads. Similarly, random-primed labeling ornick-translation labeling (supra, pp. 640-643) can also be used. In someembodiments the probe or primer are synthesized with biotinylatednucleotides or biotinylated after synthesis by methods as describedherein.

In a preferred embodiment, chemical labeling (supra, pp. 6444-671) canbe used. In this embodiment, bisulfite-catalyzed transamination,sulfonation of cytosine residues, bromine activation of T, C and Gbases, periodate oxidation of RNA or carbodiimide activation of 5′phosphates can be done.

In a preferred embodiment, photochemistry or heat-activated labeling isdone (supra, p 162-166). Thus for example, aryl azides and nitrenespreferably label adenosines, and to a less extent C and T (Aslam et al.,Bioconjugation: Protein Coupling Techniques for Biomedical Sciences; NewYork, Grove's Dictionaries, 833 pp.). Psoralen or angelicin compoundscan also be used (Aslam, p 492, supra). The preferential modification ofguanine can be accomplished via intercalation of platinum complexes(Aslam, supra).

In a preferred embodiment, the target nucleic acid can be absorbed onpositively charged surfaces, such as an amine coated solid phase. Thetarget nucleic acid can be cross-linked to the surface after physicalabsorption for increased retention (e.g. PEI coating and glutaraldehydecross-linking; Aslam, supra, p. 485).

In a preferred embodiment, direct chemical attached or photocrosslinkingcan be done to attach the target nucleic acid to the solid phase, byusing direct chemical groups on the solid phase substrate. For example,carbodiimide activation of 5′ phosphates, attachment to exocyclic amineson DNA bases, and psoralen can be attached to the solid phase forcrosslinking to the DNA. Other methods of tagging and immobilizingnucleic acids are described in U.S. Ser. No. 09/931,285, filed Aug. 16,2001, which is expressly incorporated herein by reference.

Once attached to the first solid support, the target sequence, probe orprimers are amenable to analysis as described herein.

In some embodiments when degradation is the preferred method ofperforming complexity reduction, the ddTNPs or dNTPs that are addedduring the reaction confer protection from degradation (whether chemicalor enzymatic). Thus, after the assay, the degradation components areadded, and unreacted primers are degraded, leaving only the reactedprimers. Labeled protecting groups are particularly preferred; forexample, 3′-substituted-2′-dNTPs can contain anthranylic derivativesthat are fluorescent (with alkali or enzymatic treatment for removal ofthe protecting group).

In a preferred embodiment, the secondary label is a nuclease inhibitor,such as thiol NTPs. In this embodiment, the chain-terminating NTPs arechosen to render extended primers resistant to nucleases, such as3′-exonucleases. Addition of an exonuclease will digest the non-extendedprimers leaving only the extended primers to bind to the capture probeson the array. This may also be done with OLA, wherein the ligated probewill be protected but the unprotected ligation probe will be digested.

In this embodiment, suitable 3′-exonucleases include, but are notlimited to, exo I, exo III, exo VII, and 3′-5′ exophosphodiesterases.That is, treatment with single stranded nucleases (either endonucleasesor exonucleases) will effectively remove excess nucleic acid sequencesthat are non-complementary to the locus specific primer or extensionproduct (see FIG. 11). Nuclease treatment can be performed either priorto or after separation, i.e. immobilization and washing, of purifiednucleic acid targets.

Alternatively, an 3′ exonuclease may be added to a mixture of 3′ labeledbiotin/streptavidin; only the unreacted oligonucleotides will bedegraded. Following exonuclease treatment, the exonuclease and thestreptavidin can be degraded using a protease such as proteinase K. Thesurviving nucleic acids (i.e. those that were biotinylated) are thenhybridized to the array.

In a preferred embodiment the non-hybridized nucleic acids are removedby washing. In this embodiment the hybridization complexes areimmobilized on a solid support and washed under conditions sufficient toremove non-hybridized nucleic acids, i.e. non-hybridized probes andsample nucleic acids. In a particularly preferred embodiment immobilizedcomplexes are washed under conditions sufficient to remove imperfectlyhybridized complexes. That is, hybridization complexes that containmismatches are also removed in the wash steps.

A variety of hybridization or washing conditions may be used in thepresent invention, including high, moderate and low stringencyconditions; see for example Maniatis et al., Molecular Cloning: ALaboratory Manual, 2d Edition, 1989, and Short Protocols in MolecularBiology, ed. Ausubel, et al, hereby incorporated by reference. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, “Overview of principlesof hybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 3° C. forshort probes (e.g. 10 to 50 nucleotides) and at least about 6° C. forlong probes (e.g. greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of helix destabilizing agents such asformamide. The hybridization or washing conditions may also vary when anon-ionic backbone, i.e. PNA is used, as is known in the art. Inaddition, cross-linking agents may be added after target binding tocross-link, i.e. covalently attach, the two strands of the hybridizationcomplex.

In one embodiment the hybridization complexes are immobilized by bindingof a purification tag to the solid support. That is, a purification tagis incorporated into the hybridization complexes. Purification tags aredescribed herein and can be incorporated into hybridization complexes ina variety of ways. In one embodiment the locus specific probes containpurification tags as described herein. That is, the probe is synthesizedwith a purification tag, i.e. biotinylated nucleotides, or apurification tag is added to the probe. Thus, upon hybridization withtarget nucleic acids, immobilization of the hybridization complexes isaccomplished by a purification tag. The purification tag associates withthe solid support.

Purification tags are described herein. In a preferred embodiment thepurification tag is biotin. That is, preferably the first probe islabeled with biotin. The labeled hybridization complex, therefore, bindsto streptavidin coated solid support. Solid supports also are describedherein. In a preferred embodiment the solid support is streptavidincoated magnetic beads.

The purification tag also can be incorporated into the locus specificprimer following a primer extension reaction as described more fullybelow. Briefly, following hybridization of locus specific primers withtarget nucleic acids, a polymerase extension reaction is performed. Inthis embodiment tagged nucleotides, i.e. biotinylated nucleotides, areincorporated into the primer as a result of the extension reaction. Thatis, once the target sequence and the first probe sequence havehybridized, the method of this embodiment further comprises the additionof a polymerase and at least one nucleotide (dNTP) labeled with apurification tag. Suitable DNA polymerases include, but are not limitedto, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNA polymerase. Inthis embodiment, it also is important to anneal under high stringencyconditions so that only correctly hybridized probes and target nucleicacids are extended.

In addition, the purification tag can be incorporated into the targetnucleic acid. In this embodiment, the target nucleic acid is labeledwith a purification tag and immobilized to the solid support asdescribed above. Preferably the tag is biotin.

Once formed, the tagged extension product is immobilized on the solidsupport as described above. Once immobilized, the complexes are washedso as to remove unhybridized nucleic acids.

Thus, a complexity reduction includes a locus specific selection oftarget nucleic acids. Non-specific or non-target nucleic acids areremoved.

Once unhybridized probes and non-target nucleic acids have been removed,the probes, primers or hybridization complexes are generally subjectedto an extension reaction. As outlined herein, the probes, primers orhybridization complexes can be immobilized or in solution after theoptional complexity reduction step. Using the hybridized locus specificor allele specific probe as a primer, extension enzyme such as apolymerase and dNTPs are added to the assay mixture for extension of theprimer. The resulting extended primer thus includes sequence informationof the target nucleic acid, including the sequence of the specificallele to be detected. Thus, the extended primer serves as the templatein subsequent specificity steps to identify the nucleotide at thedetection position, i.e. the particular allele to be detected.

By “extension enzyme” herein is meant an enzyme that will extend asequence by the addition of NTPs. As is well known in the art, there area wide variety of suitable extension enzymes, of which polymerases (bothRNA and DNA, depending on the composition of the target sequence andprecircle probe) are preferred. Preferred polymerases are those thatlack strand displacement activity, such that they will be capable ofadding only the necessary bases at the end of the probe, without furtherextending the probe to include nucleotides that are complementary to atargeting domain and thus preventing circularization. Suitablepolymerases include, but are not limited to, both DNA and RNApolymerases, including the Klenow fragment of DNA polymerase I,SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase,Phi29 DNA polymerase and various RNA polymerases such as from Thermussp., or Q beta replicase from bacteriophage, also SP6, T3, T4 and T7 RNApolymerases can be used, among others.

Even more preferred polymerases are those that are essentially devoid ofa 5′ to 3′ exonuclease activity, so as to assure that the probe will notbe extended past the 5′ end of the probe. Exemplary enzymes lacking 5′to 3′ exonuclease activity include the Klenow fragment of the DNAPolymerase and the Stoffel fragment of DNAPTaq Polymerase. For example,the Stoffel fragment of Taq DNA polymerase lacks 5′ to 3′ exonucleaseactivity due to genetic manipulations, which result in the production ofa truncated protein lacking the N-terminal 289 amino acids. (See e.g.,Lawyer et al., J. Biol. Chem., 264:6427-6437 [1989]; and Lawyer et al.,PCR Meth. Appl., 2:275-287 [1993]). Analogous mutant polymerases havebeen generated for polymerases derived from T. maritima, Tsps17, TZ05,Tth and Taf.

Even more preferred polymerases are those that lack a 3′ to 5′exonuclease activity, which is commonly referred to as a proof-readingactivity, and which removes bases which are mismatched at the 3′ end ofa primer-template duplex. Although the presence of 3′ to 5′ exonucleaseactivity provides increased fidelity in the strand synthesized, the 3′to 5′ exonuclease activity found in thermostable DNA polymerases such asTma (including mutant forms of Tma that lack 5′ to 3′ exonucleaseactivity) also degrades single-stranded DNA such as the primers used inthe PCR, single-stranded templates and single-stranded PCR products. Theintegrity of the 3′ end of an oligonucleotide primer used in a primerextension process is critical as it is from this terminus that extensionof the nascent strand begins. Degradation of the 3′ end leads to ashortened oligonucleotide which in turn results in a loss of specificityin the priming reaction (i.e., the shorter the primer the more likely itbecomes that spurious or non-specific priming will occur).

Yet even more preferred polymerases are thermostable polymerases. Forthe purposes of this invention, a heat resistant enzyme is defined asany enzyme that retains most of its activity after one hour at 40° C.under optimal conditions. Examples of thermostable polymerase which lackboth 5′ to 3′ exonuclease and 3′ to 5′ exonuclease include Stoffelfragment of Taq DNA polymerase. This polymerase lacks the 5′ to 3′exonuclease activity due to genetic manipulation and no 3′ to 5′activity is present as Taq polymerase is naturally lacking in 3′ to 5′exonuclease activity. Tth DNA polymerase is derived form Thermusthermophilus, and is available form Epicentre Technologies, MolecularBiology Resource Inc., or Perkin-Elmer Corp. Other useful DNApolymerases which lack 3′ exonuclease activity include a Vent[R](exo−),available from New England Biolabs, Inc., (purified from strains of E.coli that carry a DNA polymerase gene from the archaebacteriumThermococcus litoralis), and Hot Tub DNA polymerase derived from Thermusflavus and available from Amersham Corporation.

Other preferred enzymes which are thermostable and deprived of 5′ to 3′exonuclease activity and of 3′ to 5′ exonuclease activity includeAmpliTaq Gold. Other DNA polymerases, which are at least substantiallyequivalent may be used like other N-terminally truncated Thermusaquaticus (Taq) DNA polymerase I. the polymerase named KlenTaq I andKlenTaq LA are quite suitable for that purpose. Of course, any otherpolymerase having these characteristics can also be used according tothe invention.

The conditions for performing the addition of one or more nucleotides atthe 3′ end of the probe will depend on the particular enzyme used, andwill generally follow the conditions recommended by the manufacturer ofthe enzymes used.

In addition, it will be appreciated that more than one complexityreduction step can be performed. That is, following a first complexityreduction step, either the remaining target nucleic acid or the extendedlocus or allele specific primer, when applicable, are subjected to asubsequent complexity reduction step as described above. That is, anadditional locus specific or allele specific primer is hybridized to thetarget nucleic acid, which can be either the original target nucleicacid or the extended primer, and unhybridized target nucleic acids areremoved. This can be repeated as many times as necessary to achieve therequired level of enrichment of target nucleic acid.

While the above has been described in the context of complexityreduction, it is appreciated that some level of specificity also isincluded in these steps. That is, as a result of hybridizing targetnucleic acids with locus specific probes, specificity also inaccomplished. This is particularly apparent when allele specific probesare used initially.

Specificity Component

Generally following at least one complexity reduction step a specificitystep is included in the method of the invention. By “specificitycomponent” is meant a step that discriminates between target nucleicacids, preferably at the level of the allele. That is, the specificitycomponent is an allele specific step (e.g. genotyping or SNP analysis).While some level of specificity can be accomplished by simplyhybridizing allele specific probes to the template (i.e. the product ofthe complexity reduction step above), in a preferred embodiment thespecificity step includes an enzymatic step. That is, the fidelity of anenzymatic step improves specificity for allele discrimination. Preferredenzymes include DNA polymerases, RNA polymerases and ligases asdescribed in more detail herein.

Polymerases are described above. Many ligases are known and are suitablefor use in the invention, e.g. Lehman, Science, 186: 790-797 (1974);Engler et al, DNA Ligases, pages 3-30 in Boyer, editor, The Enzymes,Vol. 15B (Academic Press, New York, 1982); and the like. Preferredligases include T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taqligase, Pfu ligase, and Tth ligase. Protocols for their use are wellknown, e.g. Sambrook et al (cited above); Barany, PCR Methods anApplications, 1: 5-16 (1991); Marsh et al, Strategies, 5: 73-76 (1992);and the like. Generally, ligases require that a 5′ phosphate group bepresent for ligation to the 3′ hydroxyl of an abutting strand. Preferredligases include thermostable or (thermophilic) ligases, such as pfuligase, Tth ligase, Taq ligase and Ampligase™ DNA ligase (EpicentreTechnologies, Madison, Wis.). Ampligase has a low blunt end ligationactivity.

The preferred ligase is one which has the least mismatch ligation. Thespecificity of ligase can be increased by substituting the more specificNAD+-dependant ligases such as E. coli ligase and (thermostable) Taqligase for the less specific T4 DNA ligase. The use of NAD analogues inthe ligation reaction further increases specificity of the ligationreaction. See, U.S. Pat. No. 5,508,179 to Wallace et al.

In one embodiment the specificity component is performed withimmobilized targets. That is, the products of the complexity reductionstep are immobilized on a solid support as outlined herein and describedin U.S. Ser. No. 09/931,285, filed Aug. 16, 2001, which is expresslyincorporated herein by reference. As discussed herein the target ofspecificity reaction is referred to as a “specificity target”. That is,the product of the complexity reduction step is the specificity target.

In one embodiment the support is the same support as in the initialcomplexity reduction step. In this embodiment the target nucleic acid isremoved from the solid support prior to the specificity assay. Thetarget nucleic acid can be removed by any method that denatures thehybridization complex resulting in release of the target nucleic acid.As one of skill in the art appreciates, in this embodiment the targetnucleic acid is not covalently bound to the solid support. That is, itis the target probe that is stably attached to the support. That is,while the attachment of the probe is not necessarily covalent, it isstable enough to withstand denaturation of the hybridization complex andremoval of the non-attached target nucleic acid.

In an alternative embodiment the specificity target is in solution. Thatis, following a complexity reduction step, the hybridization complexbetween the immobilized target nucleic acid and target probe, which hasgenerally been modified (see above), is denatured and the modifiedtarget probe is eluted from the hybridization complex. In a preferredembodiment the specificity target is analyzed in solution. In analternative embodiment the solution phase specificity target isimmobilized on a subsequent solid support.

These specificity assays, i.e. genotyping techniques, fall into fivegeneral categories: (1) techniques that rely on traditionalhybridization methods that utilize the variation of stringencyconditions (temperature, buffer conditions, etc.) to distinguishnucleotides at the detection position; (2) extension techniques that adda base (“the base”) to basepair with the nucleotide at the detectionposition; (3) ligation techniques, that rely on the specificity ofligase enzymes (or, in some cases, on the specificity of chemicaltechniques), such that ligation reactions occur preferentially ifperfect complementarity exists at the detection position; (4) cleavagetechniques, that also rely on enzymatic or chemical specificity suchthat cleavage occurs preferentially if perfect complementarity exists;and (5) techniques that combine these methods. See generally WO00/63437, incorporated by reference in its entirety.

Competitive Hybridization

In a preferred embodiment, the use of competitive hybridization isperformed to elucidate either the identity of the nucleotide(s) at thedetection position or the presence of a mismatch. For example,sequencing by hybridization has been described (Drmanac et al., Genomics4:114 (1989); Koster et al., Nature Biotechnology 14:1123 (1996); U.S.Pat. Nos. 5,525,464; 5,202,231 and 5,695,940, among others, all of whichare hereby expressly incorporated by reference in their entirety).

It should be noted in this context that “mismatch” is a relative termand meant to indicate a difference in the identity of a base at aparticular position, termed the “detection position” herein, between twosequences. In general, sequences that differ from wild type sequencesare referred to as mismatches. However, particularly in the case ofSNPs, what constitutes “wild type” may be difficult to determine asmultiple alleles can be relatively frequently observed in thepopulation, and thus “mismatch” in this context requires the artificialadoption of one sequence as a standard. Thus, for the purposes of thisinvention, sequences are referred to herein as “match” and “mismatch”.Thus, the present invention may be used to detect substitutions,insertions or deletions as compared to a wild-type sequence.

In a preferred embodiment, a plurality of probes (sometimes referred toherein as “readout probes”) are used to identify the base at thedetection position. In this embodiment, each different readout probecomprises a different detection label (which, as outlined below, can beeither a primary label or a secondary label) and a different base at theposition that will hybridize to the detection position of the targetsequence (herein referred to as the readout position) such thatdifferential hybridization will occur. That is, all other parametersbeing equal, a perfectly complementary readout probe (a “match probe”)will in general be more stable and have a slower off rate than a probecomprising a mismatch (a “mismatch probe”) at any particulartemperature. Accordingly, by using different readout probes, each with adifferent base at the readout position and each with a different label,the identification of the base at the detection position is elucidated.

Accordingly, in some embodiments a detectable label is incorporated intothe readout probe. In a preferred embodiment, a set of readout probesare used, each comprising a different base at the readout position. Insome embodiments, each readout probe comprises a different label, thatis distinguishable from the others. For example, a first label may beused for probes comprising adenosine at the readout position, a secondlabel may be used for probes comprising guanine at the readout position,etc. In a preferred embodiment, the length and sequence of each readoutprobe is identical except for the readout position, although this neednot be true in all embodiments.

The number of readout probes used will vary depending on the end use ofthe assay. For example, many SNPs are biallelic, and thus two readoutprobes, each comprising an interrogation base that will basepair withone of the detection position bases. For sequencing, for example, forthe discovery of SNPs, a set of four readout probes are used, althoughSNPs may also be discovered with fewer readout parameters.

As will be appreciated by those in the art and additionally outlinedbelow, this system can take on a number of different configurations,including a solution phase assay and a solid phase assay.

Solution Phase Assay

In some embodiments a solution phase assay is performed followed byattaching the target sequence to a solid support such as an array. Afterthe competitive hybridization has occurred, the target sequence is addedto the support, which may take on several configurations, outlinedbelow.

Solid Phase Assay

In a preferred embodiment, the competition reaction is done on a solidsupport, such as an array. This system may take on severalconfigurations.

In a preferred embodiment, a sandwich assay of sorts is used. In thisembodiment, the bead, when bead arrays are used, comprises a captureprobe that will hybridize to a first target domain of a target sequence,and the readout probe will hybridize to a second target domain. In thisembodiment, the first target domain may be either unique to the target,or may be an exogeneous adapter sequence added to the target sequence asoutlined below, for example through the use of PCR reactions. Similarly,a sandwich assay is performed that utilizes a capture extender probe, asdescribed below, to attach the target sequence to the array.

Alternatively, the capture probe itself can be the readout probe; thatis, a plurality of microspheres are used, each comprising a captureprobe that has a different base at the readout position. In general, thetarget sequence then hybridizes preferentially to the capture probe mostclosely matched. In this embodiment, either the target sequence itselfis labeled (for example, it may be the product of an amplificationreaction) or a label probe may bind to the target sequence at a domainremote from the detection position. In this embodiment, since it is thelocation on the array that serves to identify the base at the detectionposition, different labels are not required.

Stringency Variation

In a preferred embodiment, sensitivity to variations in stringencyparameters are used to determine either the identity of thenucleotide(s) at the detection position or the presence of a mismatch.As a preliminary matter, the use of different stringency conditions suchas variations in temperature and buffer composition to determine thepresence or absence of mismatches in double stranded hybrids comprisinga single stranded target sequence and a probe is well known.

With particular regard to temperature, as is known in the art,differences in the number of hydrogen bonds as a function of basepairingbetween perfect matches and mismatches can be exploited as a result oftheir different Tms (the temperature at which 50% of the hybrid isdenatured). Accordingly, a hybrid comprising perfect complementaritywill melt at a higher temperature than one comprising at least onemismatch, all other parameters being equal. (It should be noted that forthe purposes of the discussion herein, all other parameters (i.e. lengthof the hybrid, nature of the backbone (i.e. naturally occurring ornucleic acid analog), the assay solution composition and the compositionof the bases, including G-C content are kept constant). However, as willbe appreciated by those in the art, these factors may be varied as well,and then taken into account.)

In general, as outlined herein, high stringency conditions are thosethat result in perfect matches remaining in hybridization complexes,while imperfect matches melt off. Similarly, low stringency conditionsare those that allow the formation of hybridization complexes with bothperfect and imperfect matches. High stringency conditions are known inthe art; see for example Maniatis et al., Molecular Cloning: ALaboratory Manual, 2d Edition, 1989, and Short Protocols in MolecularBiology, ed. Ausubel, et al., both of which are hereby incorporated byreference. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, “Overview of principles of hybridization and the strategy ofnucleic acid assays” (1993). Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 3° C. for short probes (e.g. 10 to 50nucleotides) and at least about 6° C. for long probes (e.g. greater than50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In anotherembodiment, less stringent hybridization conditions are used; forexample, moderate or low stringency conditions may be used, as are knownin the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

As will be appreciated by those in the art, mismatch detection usingtemperature may proceed in a variety of ways, and is similar to the useof readout probes as outlined above. Again, as outlined above, aplurality of readout probes may be used in a sandwich format; in thisembodiment, all the probes may bind at permissive, low temperatures(temperatures below the Tm of the mismatch); however, repeating theassay at a higher temperature (above the Tm of the mismatch) only theperfectly matched probe may bind. Thus, this system may be run withreadout probes with different detectable labels, as outlined above.Alternatively, a single probe may be used to query whether a particularbase is present.

Alternatively, as described above, the capture probe may serve as thereadout probe; in this embodiment, a single label may be used on thetarget; at temperatures above the Tm of the mismatch, only signals fromperfect matches will be seen, as the mismatch target will melt off.

Similarly, variations in buffer composition may be used to elucidate thepresence or absence of a mismatch at the detection position. Suitableconditions include, but are not limited to, formamide concentration.Thus, for example, “low” or “permissive” stringency conditions includeformamide concentrations of 0 to 10%, while “high” or “stringent”conditions utilize formamide concentrations of ≧40%. Low stringencyconditions include NaCl concentrations of ≧1 M, and high stringencyconditions include concentrations of ≦0.3 M. Furthermore, low stringencyconditions include MgCl₂ concentrations of ≧10 mM, moderate stringencyas 1-10 mM, and high stringency conditions include concentrations of ≦1mM.

In this embodiment, as for temperature, a plurality of readout probesmay be used, with different bases in the readout position (andoptionally different labels). Running the assays under the permissiveconditions and repeating under stringent conditions will allow theelucidation of the base at the detection position.

In one embodiment, the probes used as readout probes are “MolecularBeacon” probes as are generally described in Whitcombe et al., NatureBiotechnology 17:804 (1999), hereby incorporated by reference. As isknown in the art, Molecular Beacon probes form “hairpin” typestructures, with a fluorescent label on one end and a quencher on theother. In the absence of the target sequence, the ends of the hairpinhybridize, causing quenching of the label. In the presence of a targetsequence, the hairpin structure is lost in favor of target sequencebinding, resulting in a loss of quenching and thus an increase insignal.

In one embodiment, the Molecular Beacon probes can be the capture probesas outlined herein for readout probes. For example, different beadscomprising labeled Molecular Beacon probes (and different bases at thereadout position) are made optionally they comprise different labels.Alternatively, since Molecular Beacon probes can have spectrallyresolvable signals, all four probes (if a set of four different baseswith is used) differently labeled are attached to a single bead.

Extension Assays

In this embodiment the specificity target is immobilized on a solidsupport. In a preferred embodiment, extension genotyping is done. Inthis embodiment, any number of techniques are used to add a nucleotideto the readout position of a probe hybridized to the target sequenceadjacent to the detection position. By relying on enzymatic specificity,preferentially a perfectly complementary base is added. All of thesemethods rely on the enzymatic incorporation of nucleotides at thedetection position. This may be done using chain terminating dNTPs, suchthat only a single base is incorporated (e.g. single base extensionmethods), or under conditions that only a single type of nucleotide isadded followed by identification of the added nucleotide (extension andpyrosequencing techniques).

Single Base Extension

In a preferred embodiment, single base extension (SBE; sometimesreferred to as “minisequencing”) is used to determine the identity ofthe base at the detection position. SBE utilizes an extension primerthat may have at least one adapter sequence that hybridizes to thetarget nucleic acid immediately adjacent to the detection position, toform a hybridization complex. A polymerase (generally a DNA polymerase)is used to extend the 3′ end of the primer with a nucleotide ornucleotide analog. In some embodiments the nucleotide or nucleotideanalog is labeled with a detection label as described herein. Based onthe fidelity of the enzyme, a nucleotide is only incorporated into thereadout position of the growing nucleic acid strand if it is perfectlycomplementary to the base in the target strand at the detectionposition. The nucleotide may be derivatized such that no furtherextensions can occur, so only a single nucleotide is added. Once thelabeled nucleotide is added, detection of the label proceeds as outlinedherein. Again, amplification in this case is accomplished throughcycling or repeated rounds of reaction/elution, although in someembodiments amplification is not necessary. Alternatively, in someembodiments, amplification is performed prior to the extension reaction.Alternatively, amplification is performed following the extensionreaction.

The reaction is initiated by introducing the hybridization complexcomprising the specificity target on the support to a solutioncomprising a first nucleotide. In some embodiments, the nucleotidescomprise a detectable label, which may be either a primary or asecondary label. In addition, the nucleotides may be nucleotide analogs,depending on the configuration of the system. For example, if the dNTPsare added in sequential reactions, such that only a single type of dNTPcan be added, the nucleotides need not be chain terminating. Inaddition, in this embodiment, the dNTPs may all comprise the same typeof label.

Alternatively, if the reaction comprises more than one dNTP, the dNTPsshould be chain terminating, that is, they have a blocking or protectinggroup at the 3′ position such that no further dNTPs may be added by theenzyme. As will be appreciated by those in the art, any number ofnucleotide analogs may be used, as long as a polymerase enzyme willstill incorporate the nucleotide at the readout position. Preferredembodiments utilize dideoxy-triphosphate nucleotides (ddNTPs) andhalogenated dNTPs. Generally, a set of nucleotides comprising ddATP,ddCTP, ddGTP and ddTTP is used, each with a different detectable label,although as outlined herein, this may not be required. Alternativepreferred embodiments use a cyclo nucleotides (NEN). These chainterminating nucleotide analogs are particularly good substrates for Deepvent (exo⁻) and thermosequenase.

In addition, as will be appreciated by those in the art, the single baseextension reactions of the present invention allow the preciseincorporation of modified bases into a growing nucleic acid strand.Thus, any number of modified nucleotides may be incorporated for anynumber of reasons, including probing structure-function relationships(e.g. DNA:DNA or DNA:protein interactions), cleaving the nucleic acid,crosslinking the nucleic acid, incorporate mismatches, etc.

As will be appreciated by those in the art, the configuration of thegenotyping SBE system can take on several forms.

Multi-Base Extension

In a preferred embodiment genotyping is accomplished by primer extensionthat does not use chain terminating nucleotides. As such, thisgenotyping is considered multi-base extension. The method includesproviding an interrogator oligonucleotide designed to detect one alleleof a given SNP. The number of oligonucleotides is determined by thenumber of distinct SNP alleles being probed. For instance, if one wereprobing 1000 SNPs, each with two alleles, 2000 oligonucleotides would benecessary. The interrogators are complementary to a stretch of DNAcontaining the SNP, with the terminal base of each interrogatorcorresponding to the SNP position, or with the SNP-specific positionwithin the last 1, 2 3 or 4 nucleotides of the interrogator. In somepreferred embodiments the interrogator is not the terminal position ofthe primer, but rather resides at a position 1, 2, 3, 4, 5 or 6nucleotides from the 3′ terminus of the primer. For example, when a SNPhas an A and C allele, interrogators ending in T and G are provided andin some embodiments may be immobilized on separate elements (beads) todetect the two. Although both the match and the mismatch will hybridizeto a given allele, only the match can act as a primer for a DNApolymerase extension reaction. Accordingly, following hybridization ofthe probes with the target DNA, a polymerase reaction is performed. Thisresults in the extension of the hybrids with a DNA polymerase in thepresence of labeled dNTPs. The labeled dNTPs are selectivelyincorporated into the extension product that results from the probe thatis complementary to the SNP position.

In one embodiment, address oligonucleotides (adapters) are incorporatedinto the interrogator oligonucleotides. As such, in one embodiment oneperforms the hybridization and extension steps in fluid phase in theabsence of beads. Each allele contains a unique adapter. Afterhybridization/extension the products are hybridized to an array ofcomplementary address sequences for signal detection and analysis.

Solution Phase Assay

As for the OLA reaction described below, the reaction may be done insolution, and then the newly synthesized strands, with the base-specificdetectable labels, can be detected. For example, they can be directlyhybridized to capture probes that are complementary to the extensionprimers, and the presence of the label is then detected. As will beappreciated by those in the art, a preferred embodiment utilizes fourdifferent detectable labels, i.e. one for each base, such that uponhybridization to the capture probe on the array, the identification ofthe base can be done isothermally.

In a preferred embodiment, adapter sequences can be used in a solutionformat. In this embodiment, a single label can be used with a set offour separate primer extension reactions. In this embodiment, theextension reaction is done in solution; each reaction comprises adifferent dNTP with the label or labeled ddNTP when chain termination isdesired. For each locus genotyped, a set of four different extensionprimers are used, each with a portion that will hybridize to the targetsequence, a different readout base and each with a different adaptersequence of 15-40 bases, as is more fully outlined below. After theprimer extension reaction is complete, the four separate reactions arepooled and hybridized to an array comprising complementary probes to theadapter sequences. A genotype is derived by comparing the probeintensities of the four different hybridized adapter sequencescorresponding to a given locus.

In addition, since unextended primers do not comprise labels, theunextended primers need not be removed. However, they may be, ifdesired, as outlined below; for example, if a large excess of primersare used, there may not be sufficient signal from the extended primerscompeting for binding to the surface.

Alternatively, one of skill in the art could use a single label andtemperature to determine the identity of the base; that is, the readoutposition of the extension primer hybridizes to a position on the captureprobe. However, since the three mismatches will have lower Tms than theperfect match, the use of temperature could elucidate the identity ofthe detection position base.

Solid Phase Assay

Alternatively, the reaction may be done on a surface by capturing thetarget sequence and then running the SBE reaction, in a sandwich typeformat. In this embodiment, the capture probe hybridizes to a firstdomain of the target sequence (which can be endogeneous or an exogeneousadapter sequence added during an amplification reaction), and theextension primer hybridizes to a second target domain immediatelyadjacent to the detection position. The addition of the enzyme and therequired NTPs results in the addition of the interrogation base. In thisembodiment, each NTP must have a unique label. Alternatively, each NTPreaction may be done sequentially on a different array. As is known byone of skill in the art, ddNTP and dNTP are the preferred substrateswhen DNA polymerase is the added enzyme; NTP is the preferred substratewhen RNA polymerase is the added enzyme.

Furthermore, capture extender probes can be used to attach the targetsequence to the bead. In this embodiment, the hybridization complexcomprises the capture probe, the target sequence and the adaptersequence.

Similarly, the capture probe itself can be used as the extension probe,with its terminus being directly adjacent to the detection position.Upon the addition of the target sequence and the SBE reagents, themodified primer is formed comprising a detectable label, and thendetected. Again, as for the solution based reaction, each NTP must havea unique label, the reactions must proceed sequentially, or differentarrays must be used. Again, as is known by one of skill in the art,ddNTP and dNTP are the preferred substrates when DNA polymerase is theadded enzyme; NTP is the preferred substrate when RNA polymerase is theadded enzyme.

In addition, as outlined herein, the target sequence may be directlyattached to the array; the extension primer hybridizes to it and thereaction proceeds.

Variations on this include, where the capture probe and the extensionprobe adjacently hybridize to the target sequence. Either before orafter extension of the extension probe, a ligation step may be used toattach the capture and extension probes together for stability. Theseare further described below as combination assays.

As will be appreciated by those in the art, the determination of thebase at the detection position can proceed in several ways. In apreferred embodiment, the reaction is run with all four nucleotides(assuming all four nucleotides are required), each with a differentlabel, as is generally outlined herein. Alternatively, a single label isused, by using four reactions In a preferred embodiment, universalprimers or adapters specific for the nucleotide at a detection positionare used and detected as outlined below.

Removal of Unextended Primers

In a preferred embodiment, for both SBE as well as a number of otherreactions outlined herein, it is desirable to remove the unextended orunreacted primers from the assay mixture, and particularly from thearray, as unextended primers will compete with the extended (labeled)primers in binding to capture probes, thereby diminishing the signal.The concentration of the unextended primers relative to the extendedprimer may be relatively high, since a large excess of primer is usuallyrequired to generate efficient primer annealing. Accordingly, a numberof different techniques may be used to facilitate the removal ofunextended primers. As outlined above, these generally include methodsbased on removal of unreacted primers by binding to a solid support,protecting the reacted primers and degrading the unextended ones, andseparating the unreacted and reacted primers.

Separation Systems

The use of secondary label systems (and even some primary label systems)can be used to separate unreacted and reacted probes; for example, theaddition of streptavidin to a nucleic acid greatly increases its size,as well as changes its physical properties, to allow more efficientseparation techniques. For example, the mixtures can be sizefractionated by exclusion chromatography, affinity chromatography,filtration or differential precipitation.

Non-Terminated Extension

In a preferred embodiment, methods of adding a single base are used thatdo not rely on chain termination. That is, similar to SBE, enzymaticreactions that utilize dNTPs and polymerases can be used; however,rather than use chain terminating dNTPs, regular dNTPs are used. Thismethod relies on a time-resolved basis of detection; only one type ofbase is added during the reaction.

Pyrosequencing

Pyrosequencing is an extension and sequencing method that can be used toadd one or more nucleotides to the detection position(s); it is verysimilar to SBE except that chain terminating NTPs need not be used(although they may be). Pyrosequencing relies on the detection of areaction product, PPi, produced during the addition of an NTP to agrowing oligonucleotide chain, rather than on a label attached to thenucleotide. One molecule of PPi is produced per dNTP added to theextension primer. That is, by running sequential reactions with each ofthe nucleotides, and monitoring the reaction products, the identity ofthe added base is determined.

The release of pyrophosphate (PPi) during the DNA polymerase reactioncan be quantitatively measured by many different methods and a number ofenzymatic methods have been described; see Reeves et al., Anal. Biochem.28:282 (1969); Guillory et al., Anal. Biochem. 39:170 (1971); Johnson etal., Anal. Biochem. 15:273 (1968); Cook et al., Anal. Biochem. 91:557(1978); Drake et al., Anal. Biochem. 94:117 (1979); WO93/23564; WO98/28440; WO98/13523; Nyren et al., Anal. Biochem. 151:504 (1985); allof which are incorporated by reference. The latter method allowscontinuous monitoring of PPi and has been termed ELIDA (EnzymaticLuminometric Inorganic Pyrophosphate Detection Assay). A preferredembodiment utilizes any method which can result in the generation of anoptical signal, with preferred embodiments utilizing the generation of achemiluminescent or fluorescent signal.

A preferred method monitors the creation of PPi by the conversion of PPito ATP by the enzyme sulfurylase, and the subsequent production ofvisible light by firefly luciferase (see Ronaghi et al., Science 281:363(1998), incorporated by reference). In this method, the fourdeoxynucleotides (dATP, dGTP, dCTP and dTTP; collectively dNTPs) areadded stepwise to a partial duplex comprising a sequencing primerhybridized to a single stranded DNA template and incubated with DNApolymerase, ATP sulfurylase, luciferase, and optionally anucleotide-degrading enzyme such as apyrase. A dNTP is only incorporatedinto the growing DNA strand if it is complementary to the base in thetemplate strand. The synthesis of DNA is accompanied by the release ofPPi equal in molarity to the incorporated dNTP. The PPi is converted toATP and the light generated by the luciferase is directly proportionalto the amount of ATP. In some cases the unincorporated dNTPs and theproduced ATP are degraded between each cycle by the nucleotide degradingenzyme.

Accordingly, a preferred embodiment of the methods of the invention isas follows. A substrate comprising the target sequences and extensionprimers, forming hybridization complexes, is dipped or contacted with areaction volume (chamber or well) comprising a single type of dNTP, anextension enzyme, and the reagents and enzymes necessary to detect PPi.If the dNTP is complementary to the base of the target portion of thetarget sequence adjacent to the extension primer, the dNTP is added,releasing PPi and generating detectable light, which is detected asgenerally described in U.S. Ser. Nos. 09/151,877 and 09/189,543, and PCTUS98/09163, all of which are hereby incorporated by reference. If thedNTP is not complementary, no detectable signal results. The substrateis then contacted with a second reaction volume (chamber) comprising adifferent dNTP and the additional components of the assay. This processis repeated if the identity of a base at a second detection position isdesirable.

In a preferred embodiment, washing steps may be done in between the dNTPreactions, as required. These washing steps may optionally comprise anucleotide-degrading enzyme, to remove any unreacted dNTP and decreasingthe background signal, as is described in WO 98/28440, incorporatedherein by reference.

As will be appreciated by those in the art, the system can be configuredin a variety of ways, including both a linear progression or a circularone; for example, four arrays may be used that each can dip into one offour reaction chambers arrayed in a circular pattern. Each cycle ofsequencing and reading is followed by a 90 degree rotation, so that eachsubstrate then dips into the next reaction well.

As for simple extension and SBE, the pyrosequencing systems may beconfigured in a variety of ways; for example, the target sequence may beimmobilized in a variety of ways, including direct attachment of thetarget sequence; the use of a capture probe with a separate extensionprobe; the use of a capture extender probe, a capture probe and aseparate extension probe; the use of adapter sequences in the targetsequence with capture and extension probes; and the use of a captureprobe that also serves as the extension probe.

One additional benefit of pyrosequencing for genotyping purposes is thatsince the reaction does not rely on the incorporation of labels into agrowing chain, the unreacted extension primers need not be removed.

In addition, pyrosequencing can be used as a “switch” to activate adetectable enzymatic reaction, thus providing an amplification of sorts.The by-product of the polymerase reaction, PPi, is converted to ATPduring pyrosequencing reactions. In standard pyrosequencing thatutilizes a luciferase/luciferin assay, the detection sensitivity islimited because only a single photon is generated per nucleotideincorporation event. However, in a preferred embodiment, if PPi, or asimple enzymatic derivative such as Pi or ATP is used to “activate” anenzyme or protein, the detection sensitivity is increased. A number ofdifferent proteins are either “on” or “off” depending on theirphosphorylation status. In this was, PPi (or ATP) acts a “switch” toturn on or off a stream of detection molecules, similar to the way atransistor controls a large flow of electricity by using a small currentor potential to gat the process. That is, the generation of PPi resultsin an enzymatic cascade that results in a detectable event; the PPigeneration results in a “switch”. For example, ATP may be used tophosphorylate a peroxidase enzyme, which when phosphorylated becomes“active” like horse radish peroxidase (HRP). This HRP activity is thendetected using standar hydrogen peroxide/luminol HRP detection systems.There are a large number of enzymes and proteins regulated byphosphorylation. What is important is that the activating or switchenzyme that utilizes Pi, PPi or ATP as the substrate discriminates theactivating species from the original dNTP used in the extensionreaction.

Allelic PCR

In a preferred embodiment, the method used to detect the base at thedetection position is allelic PCR, referred to herein as “aPCR”. Asdescribed in Newton et al., Nucl. Acid Res. 17:2503 (1989), herebyexpressly incorporated by reference, allelic PCR allows single basediscrimination based on the fact that the PCR reaction does not proceedwell if the terminal 3′-nucleotide is mismatched, assuming the DNApolymerase being used lacks a 3′-exonuclease proofreading activity.Accordingly, the identification of the base proceeds by using allelicPCR primers (sometimes referred to herein as aPCR primers) that havereadout positions at their 3′ ends. Thus the target sequence comprises afirst domain comprising at its 5′ end a detection position.

In general, aPCR may be briefly described as follows. A double strandedtarget nucleic acid is denatured, generally by raising the temperature,and then cooled in the presence of an excess of a aPCR primer, whichthen hybridizes to the first target strand. If the readout position ofthe aPCR primer basepairs correctly with the detection position of thetarget sequence, a DNA polymerase (again, that lacks 3′-exonucleaseactivity) then acts to extend the primer with dNTPs, resulting in thesynthesis of a new strand forming a hybridization complex. The sample isthen heated again, to disassociate the hybridization complex, and theprocess is repeated. By using a second PCR primer for the complementarytarget strand, rapid and exponential amplification occurs. Thus aPCRsteps are denaturation, annealing and extension. The particulars of aPCRare well known, and include the use of a thermostable polymerase such asTaq I polymerase and thermal cycling.

Accordingly, the aPCR reaction requires at least one aPCR primer, apolymerase, and a set of dNTPs. As outlined herein, the primers maycomprise the label, or one or more of the dNTPs may comprise a label.

Furthermore, the aPCR reaction may be run as a competition assay ofsorts. For example, for biallelic SNPs, a first aPCR primer comprising afirst base at the readout position and a first label, and a second aPCRprimer comprising a different base at the readout position and a secondlabel, may be used. The PCR primer for the other strand is the same. Theexamination of the ratio of the two colors can serve to identify thebase at the detection position.

Allelic Primer Extension

In this embodiment allele specific primers when hybridized with theircomplementary target sequence serve as template for primer extensionwith a DNA polymerase. In some respects the method is similar to aPCR asdescribed herein with the exception that only one primer need hybridizewith the target sequence prior to amplification. That is, in contrastwith PCR amplification that requires two primers, only one primer isnecessary for amplification according to the method.

In a preferred embodiment, the primer is immobilized. In a preferredembodiment the primer is immobilized to microspheres or beads asdescribed herein.

In general, as is more fully outlined below, the capture probes on thebeads of the array are designed to be substantially complementary to theextended part of the primer; that is, unextended primers will not bindto the capture probes.

Ligation Techniques for Genotyping

In this embodiment, the readout of the base at the detection positionproceeds using a ligase. In this embodiment, it is the specificity ofthe ligase which is the basis of the genotyping; that is, ligasesgenerally require that the 5′ and 3′ ends of the ligation probes haveperfect complementarity to the target for ligation to occur. Thus, in apreferred embodiment, the identity of the base at the detection positionproceeds utilizing OLA as described above. The method can be run atleast two different ways; in a first embodiment, only one strand of atarget sequence is used as a template for ligation; alternatively, bothstrands may be used; the latter is generally referred to as LigationChain Reaction or LCR.

This method is based on the fact that two probes can be preferentiallyligated together, if they are hybridized to a target strand and ifperfect complementarity exists at the two bases being ligated together.Thus, in this embodiment, the target sequence comprises a contiguousfirst target domain comprising the detection position and a secondtarget domain adjacent to the detection position. That is, the detectionposition is “between” the rest of the first target domain and the secondtarget domain, or the detection position is one nucleotide from the 3′terminus of one of the ligation probes. A first ligation probe ishybridized to the first target domain and a second ligation probe ishybridized to the second target domain. If the first ligation probe hasa base perfectly complementary to the detection position base, and theadjacent base on the second probe has perfect complementarity to itsposition, a ligation structure is formed such that the two probes can beligated together to form a ligated probe. If this complementarity doesnot exist, no ligation structure is formed and the probes are notligated together to an appreciable degree. This may be done using heatcycling, to allow the ligated probe to be denatured off the targetsequence such that it may serve as a template for further reactions. Inaddition, as is more fully outlined below, this method may also be doneusing ligation probes that are separated by one or more nucleotides, ifdNTPs and a polymerase are added (this is sometimes referred to as“Genetic Bit” analysis).

In a preferred embodiment, LCR is done for two strands of adouble-stranded target sequence. The target sequence is denatured, andtwo sets of probes are added: one set as outlined above for one strandof the target, and a separate set (i.e. third and fourth primer probenucleic acids) for the other strand of the target. In a preferredembodiment, the first and third probes will hybridize, and the secondand fourth probes will hybridize, such that amplification can occur.That is, when the first and second probes have been attached, theligated probe can now be used as a template, in addition to the secondtarget sequence, for the attachment of the third and fourth probes.Similarly, the ligated third and fourth probes will serve as a templatefor the attachment of the first and second probes, in addition to thefirst target strand. In this way, an exponential, rather than just alinear, amplification can occur.

As will be appreciated by those in the art, the ligation product can bedetected in a variety of ways.

Preferably, detection is accomplished by removing the unligated labeledprobe from the reaction before application to a capture probe. In oneembodiment, the unligated probes are removed by digesting 3′non-protected oligonucleotides with a 3′ exonuclease, such as,exonuclease I. The ligation products are protected from exo I digestionby including, for example, the use of a number of sequentialphosphorothioate residues at their 3′ terminus (for example at leastfour), thereby, rendering them resistant to exonuclease digestion. Theunligated detection oligonucleotides are not protected and are digested.

As for most or all of the methods described herein, the assay can takeon a solution-based form or a solid-phase form.

Solution Based OLA

In a preferred embodiment, the ligation reaction is run in solution. Inthis embodiment, only one of the primers carries a detectable label,e.g. the first ligation probe, and the capture probe on the bead issubstantially complementary to the other probe, e.g. the second ligationprobe. In this way, unextended labeled ligation primers will notinterfere with the assay. This substantially reduces or eliminates falsesignal generated by the optically-labeled 3′ primers.

In addition, a solution-based OLA assay that utilizes adapter sequencesmay be done. In this embodiment, rather than have the target sequencecomprise the adapter sequences, one of the ligation probes comprises theadapter sequence. This facilitates the creation of “universal arrays”.For example, the first ligation probe has an adapter sequence that isused to attach the ligated probe to the array.

Again, as outlined above for SBE, unreacted ligation primers may beremoved from the mixture as needed. For example, the first ligationprobe may comprise the label (either a primary or secondary label) andthe second may be blocked at its 3′ end with an exonuclease blockingmoiety; after ligation and the introduction of the nuclease, the labeledligation probe will be digested, leaving the ligation product and thesecond probe; however, since the second probe is unlabeled, it iseffectively silent in the assay. Similarly, the second probe maycomprise a binding partner used to pull out the ligated probes, leavingunligated labeled ligation probes behind. The binding pair is thendisassociated for subsequent amplification or detection.

Solid Phase Based OLA

Alternatively, the target nucleic acid is immobilized on a solid-phasesurface. The OLA assay is performed and unligated oligonucleotides areremoved by washing under appropriate stringency to remove unligatedoligonucleotides and thus the label. For example, the capture probe cancomprise one of the ligation probes.

Again, as outlined above, the detection of the OLA reaction can alsooccur directly, in the case where one or both of the primers comprisesat least one detectable label, or indirectly, using sandwich assays,through the use of additional probes; that is, the ligated probes canserve as target sequences, and detection may utilize amplificationprobes, capture probes, capture extender probes, label probes, and labelextender probes, etc. Alternatively, the OLA product is amplified. In apreferred embodiment the amplicons comprise labels.

In some embodiments target nucleic acids include both DNA and RNA. In apreferred embodiment RNA is mRNA. In some embodiments when RNA is thetarget nucleic acid, it is desirable to perform a reverse transcriptionassay prior to OLA as described herein. The reverse transcription assayresults in the formation of cDNA. This method is particularlyadvantageous in determining either gene expression levels or genotyping,or both. That is, the cDNA is representative of the level of mRNA.Accordingly, gene expression analysis is performed. In addition, thecDNA also serves as a template for OLA which allows for genotyping.Thus, the use of both DNA and/or RNA allows for increased multiplexingof samples on an array.

Solid Phase Oligonucleotide Ligation Assay (SPOLA)

In a preferred embodiment, a novel method of OLA is used, termed herein“solid phase oligonucleotide assay”, or “SPOLA”. In this embodiment, theligation probes are both attached to the same site on the surface of thearray (e.g. when microsphere arrays are used, to the same bead), one atits 5′ end (the “upstream probe”) and one at its 3′ end (the “downstreamprobe”). This may be done as is will be appreciated by those in the art.At least one of the probes is attached via a cleavable linker, that uponcleavage, forms a reactive or detectable (fluorophore) moiety. Ifligation occurs, the reactive moiety remains associated with thesurface; but if no ligation occurs, due to a mismatch, the reactivemoiety is free in solution to diffuse away from the surface of thearray. The reactive moiety is then used to add a detectable label.

Generally, as will be appreciated by those in the art, cleavage of thecleavable linker should result in asymmetrical products; i.e. one of the“ends” should be reactive, and the other should not, with theconfiguration of the system such that the reactive moiety remainsassociated with the surface if ligation occurred. Thus, for example,amino acids or succinate esters can be cleaved either enzymatically (viapeptidases (aminopeptidase and carboxypeptidase) or proteases) orchemically (acid/base hydrolysis) to produce an amine and a carboxylgroup. One of these groups can then be used to add a detectable label,as will be appreciated by those in the art and discussed herein.

Padlock Probe Ligation

In a preferred embodiment, the ligation probes are specialized probescalled “padlock probes”. Nilsson et al, 1994, Science 265:2085, herebyincorporated by reference. These probes have a first ligation domainthat is identical to a first ligation probe, in that it hybridizes to afirst target sequence domain, and a second ligation domain, identical tothe second ligation probe, that hybridizes to an adjacent targetsequence domain. Again, as for OLA, the detection position can be eitherat the 3′ end of the first ligation domain or at the 5′ end of thesecond ligation domain. However, the two ligation domains are connectedby a linker, frequently nucleic acid. The configuration of the system issuch that upon ligation of the first and second ligation domains of thepadlock probe, the probe forms a circular probe, and forms a complexwith the target sequence wherein the target sequence is “inserted” intothe loop of the circle.

In this embodiment, the unligated probes may be removed throughdegradation (for example, through a nuclease), as there are no “freeends” in the ligated probe.

Cleavage Techniques for Genotyping

In a preferred embodiment, the specificity for genotyping is provided bya cleavage enzyme. There are a variety of enzymes known to cleave atspecific sites, either based on sequence specificity, such asrestriction endonucleases, or using structural specificity, such as isdone through the use of invasive cleavage technology.

Endonuclease Techniques

In a preferred embodiment, enzymes that rely on sequence specificity areused. In general, these systems rely on the cleavage of double strandedsequence containing a specific sequence recognized by a nuclease,preferably an endonuclease including resolvases.

These systems may work in a variety of ways. In one embodiment, alabeled readout probe (generally attached to a bead of the array) isused; the binding of the target sequence forms a double strandedsequence that a restriction endonuclease can then recognize and cleave,if the correct sequence is present. The cleavage results in the loss ofthe label, and thus a loss of signal.

Alternatively, as will be appreciated by those in the art, a labelledtarget sequence may be used as well; for example, a labelled primer maybe used in the PCR amplification of the target, such that the label isincorporated in such a manner as to be cleaved off by the enzyme.

Alternatively, the readout probe (or, again, the target sequence) maycomprise both a fluorescent label and a quencher, as is known in theart. In this embodiment, the label and the quencher are attached todifferent nucleosides, yet are close enough that the quencher moleculeresults in little or no signal being present. Upon the introduction ofthe enzyme, the quencher is cleaved off, leaving the label, and allowingsignaling by the label.

In addition, as will be appreciated by those in the art, these systemscan be both solution-based assays or solid-phase assays, as outlinedherein.

Furthermore, there are some systems that do not require cleavage fordetection; for example, some nucleic acid binding proteins will bind tospecific sequences and can thus serve as a secondary label. For example,some transcription factors will bind in a highly sequence dependentmanner, and can distinguish between two SNPs. Having bound to thehybridization complex, a detectable binding partner can be added fordetection. In addition, mismatch binding proteins based on mutatedtranscription factors can be used.

In addition, as will be appreciated by those in the art, this type ofapproach works with other cleavage methods as well, for example the useof invasive cleavage methods, as outlined below.

Invasive Cleavage

In a preferred embodiment, the determination of the identity of the baseat the detection position of the target sequence proceeds using invasivecleavage technology. As outlined above for amplification, invasivecleavage techniques rely on the use of structure-specific nucleases,where the structure can be formed as a result of the presence or absenceof a mismatch. Generally, invasive cleavage technology may be describedas follows. A target nucleic acid is recognized by two distinct probes.A first probe, generally referred to herein as an “invader” probe, issubstantially complementary to a first portion of the target nucleicacid. A second probe, generally referred to herein as a “signal probe”,is partially complementary to the target nucleic acid; the 3′ end of thesignal oligonucleotide is substantially complementary to the targetsequence while the 5′ end is non-complementary and preferably forms asingle-stranded “tail” or “arm”. The non-complementary end of the secondprobe preferably comprises a “generic” or “unique” sequence, frequentlyreferred to herein as a “detection sequence”, that is used to indicatethe presence or absence of the target nucleic acid, as described below.The detection sequence of the second probe preferably comprises at leastone detectable label. Alternative methods have the detection sequencefunctioning as a target sequence for a capture probe, and thus rely onsandwich configurations using label probes.

Hybridization of the first and second oligonucleotides near or adjacentto one another on the target nucleic acid forms a number of structures.In a preferred embodiment, a forked cleavage structure forms and is asubstrate of a nuclease which cleaves the detection sequence from thesignal oligonucleotide. The site of cleavage is controlled by thedistance or overlap between the 3′ end of the invader oligonucleotideand the downstream fork of the signal oligonucleotide. Therefore,neither oligonucleotide is subject to cleavage when misaligned or whenunattached to target nucleic acid.

As above, the invasive cleavage assay is preferably performed on anarray format. In a preferred embodiment, the signal probe has adetectable label, attached 5′ from the site of nuclease cleavage (e.g.within the detection sequence) and a capture tag, as described hereinfor removal of the unreacted products (e.g. biotin or other hapten) 3′from the site of nuclease cleavage. After the assay is carried out, theuncleaved probe and the 3′ portion of the cleaved signal probe (e.g. thedetection sequence) may be extracted, for example, by binding tostreptavidin beads or by crosslinking through the capture tag to produceaggregates or by antibody to an attached hapten. By “capture tag” hereinis a meant one of a pair of binding partners as described above, such asantigen/antibody pairs, digoxygenenin, dinitrophenol, etc.

The cleaved 5′ region, e.g. the detection sequence, of the signal probe,comprises a label and is detected and optionally quantitated. In oneembodiment, the cleaved 5′ region is hybridized to a probe on an array(capture probe) and optically detected. As described below, manydifferent signal probes can be analyzed in parallel by hybridization totheir complementary probes in an array. In a preferred embodiment,combination techniques are used to obtain higher specificity and reducethe detection of contaminating uncleaved signal probe or incorrectlycleaved product, an enzymatic recognition step is introduced in thearray capture procedure. For example, as more fully outlined below, thecleaved signal probe binds to a capture probe to produce adouble-stranded nucleic acid in the array. In this embodiment, the 3′end of the cleaved signal probe is adjacent to the 5′ end of one strandof the capture probe, thereby, forming a substrate for DNA ligase(Broude et al. 1991. PNAS 91: 3072-3076). Only correctly cleaved productis ligated to the capture probe. Other incorrectly hybridized andnon-cleaved signal probes are removed, for example, by heatdenaturation, high stringency washes, and other methods that disruptbase pairing.

Accordingly, the present invention provides methods of determining theidentity of a base at the detection position of a target sequence. Inthis embodiment, the target sequence comprises, 5′ to 3′, a first targetdomain comprising an overlap domain comprising at least a nucleotide inthe detection position, and a second target domain contiguous with thedetection position. A first probe (the “invader probe”) is hybridized tothe first target domain of the target sequence. A second probe (the“signal probe”), comprising a first portion that hybridizes to thesecond target domain of the target sequence and a second portion thatdoes not hybridize to the target sequence, is hybridized to the secondtarget domain. If the second probe comprises a base that is perfectlycomplementary to the detection position a cleavage structure is formed.The addition of a cleavage enzyme, such as is described in U.S. Pat.Nos. 5,846,717; 5,614,402; 5,719,029; 5,541,311 and 5,843,669, all ofwhich are expressly incorporated by reference, results in the cleavageof the detection sequence from the signaling probe. This then can beused as a target sequence in an assay complex.

In addition, as for a variety of the techniques outlined herein,unreacted probes (i.e. signaling probes, in the case of invasivecleavage), may be removed using any number of techniques. For example,the use of a binding partner coupled to a solid support comprising theother member of the binding pair can be done. Similarly, after cleavageof the primary signal probe, the newly created cleavage products can beselectively labeled at the 3′ or 5′ ends using enzymatic or chemicalmethods.

Again, as outlined above, the detection of the invasive cleavagereaction can occur directly, in the case where the detection sequencecomprises at least one label, or indirectly, using sandwich assays,through the use of additional probes; that is, the detection sequencescan serve as target sequences, and detection may utilize amplificationprobes, capture probes, capture extender probes, label probes, and labelextender probes, etc.

In addition, as for most of the techniques outlined herein, thesetechniques may be done for the two strands of a double-stranded targetsequence. The target sequence is denatured, and two sets of probes areadded: one set as outlined above for one strand of the target, and aseparate set for the other strand of the target.

Thus, the invasive cleavage reaction requires, in no particular order,an invader probe, a signaling probe, and a cleavage enzyme.

As for other methods outlined herein, the invasive cleavage reaction maybe done as a solution based assay or a solid phase assay.

Solution-Based Invasive Cleavage

The invasive cleavage reaction may be done in solution, followed byaddition of one of the components to an array, with optional (butpreferable) removal of unreacted probes. For example, the reaction iscarried out in solution, using a capture tag (i.e. a member of a bindingpartner pair) that is separated from the label on the detection sequencewith the cleavage site. After cleavage (dependent on the base at thedetection position), the signaling probe is cleaved. The capture tag isused to remove the uncleaved probes (for example, using magneticparticles comprising the other member of the binding pair), and theremaining solution is added to the array. The detection sequence can bedirectly attached to the capture probe. In this embodiment, thedetection sequence can effectively act as an adapter sequence. Inalternate embodiments, the detection sequence is unlabelled and anadditional label probe is used; as outlined below, this can be ligatedto the hybridization complex.

Solid-Phase Based Assays

The invasive cleavage reaction can also be done as a solid-phase assay.The target sequence can be attached to the array using a capture probe(in addition, although not shown, the target sequence may be directlyattached to the array). In a preferred embodiment, the signaling probecomprises both a fluorophore label (attached to the portion of thesignaling probe that hybridizes to the target) and a quencher (generallyon the detection sequence), with a cleavage site in between. Thus, inthe absence of cleavage, very little signal is seen due to the quenchingreaction. After cleavage, however, the detection sequence is removed,along with the quencher, leaving the unquenched fluorophore. Similarly,the invasive probe may be attached to the array.

In a preferred embodiment, the invasive cleavage reaction is configuredto utilize a fluorophore-quencher reaction. A signaling probe comprisingboth a fluorophore and a quencher is attached to the bead. Thefluorophore is contained on the portion of the signaling probe thathybridizes to the target sequence, and the quencher is contained on aportion of the signaling probe that is on the other side of the cleavagesite (termed the “detection sequence” herein). In a preferredembodiment, it is the 3′ end of the signaling probe that is attached tothe bead (although as will be appreciated by those in the art, thesystem can be configured in a variety of different ways, includingmethods that would result in a loss of signal upon cleavage). Thus, thequencher molecule is located 5′ to the cleavage site. Upon assembly ofan assay complex, comprising the target sequence, an invader probe, anda signaling probe, and the introduction of the cleavage enzyme, thecleavage of the complex results in the disassociation of the quencherfrom the complex, resulting in an increase in fluorescence.

In this embodiment, suitable fluorophore-quencher pairs are as known inthe art. For example, suitable quencher molecules comprise Dabcyl.

Redundant Genotyping

In a preferred embodiment, the invention provides a method of increasingthe confidence of genotyping results. The method includes performinggenotyping more than once on a particular target sequence. That is, asample or target analyte is genotyped at least twice. Preferably, thesample is genotyped with different techniques such as Invader™ and OLAas described herein. If the results of the individual genotyping assaysagree, then confidence that the genotyping results are correct isincreased.

Amplification Reactions

In this embodiment, the invention provides compositions and methods foramplification and/or detection (and optionally quantification) ofproducts of nucleic acid amplification reactions. Suitable amplificationmethods include both target amplification and signal amplification.Target amplification involves the amplification (i.e. replication) ofthe target sequence to be detected, resulting in a significant increasein the number of target molecules. Target amplification strategiesinclude but are not limited to the polymerase chain reaction (PCR),strand displacement amplification (SDA), and nucleic acid sequence basedamplification (NASBA).

Alternatively, rather than amplify the target, alternate techniques usethe target as a template to replicate a signaling probe, allowing asmall number of target molecules to result in a large number ofsignaling probes, that then can be detected. Signal amplificationstrategies include the ligase chain reaction (LCR), cycling probetechnology (CPT), invasive cleavage techniques such as Invader™technology, Q-Beta replicase (QβR) technology, and the use of“amplification probes” such as “branched DNA” that result in multiplelabel probes binding to a single target sequence.

All of these methods require a primer nucleic acid (including nucleicacid analogs) that is hybridized to a target sequence to form ahybridization complex, and an enzyme is added that in some way modifiesthe primer to form a modified primer. For example, PCR generallyrequires two primers, dNTPs and a DNA polymerase; LCR requires twoprimers that adjacently hybridize to the target sequence and a ligase;CPT requires one cleavable primer and a cleaving enzyme; invasivecleavage requires two primers and a cleavage enzyme; etc. Thus, ingeneral, a target nucleic acid is added to a reaction mixture thatcomprises the necessary amplification components, and a modified primeris formed.

In general, the modified primer comprises a detectable label, such as afluorescent label, which is either incorporated by the enzyme or presenton the original primer. As required, the unreacted primers are removed,in a variety of ways, as will be appreciated by those in the art andoutlined herein. The hybridization complex is then disassociated, andthe modified primer is detected and optionally quantitated by amicrosphere array. In some cases, the newly modified primer serves as atarget sequence for a secondary reaction, which then produces a numberof amplified strands, which can be detected as outlined herein.

Accordingly, the reaction starts with the addition of a primer nucleicacid to the target sequence which forms a hybridization complex. Oncethe hybridization complex between the primer and the target sequence hasbeen formed, an enzyme, sometimes termed an “amplification enzyme”, isused to modify the primer. As for all the methods outlined herein, theenzymes may be added at any point during the assay, either prior to,during, or after the addition of the primers. The identity of the enzymewill depend on the amplification technique used, as is more fullyoutlined below. Similarly, the modification will depend on theamplification technique, as outlined below.

Once the enzyme has modified the primer to form a modified primer, thehybridization complex is disassociated. In one aspect, dissociation isby modification of the assay conditions. In another aspect, the modifiedprimer no longer hybridizes to the target nucleic acid and dissociates.Either one or both of these aspects can be employed in signal and targetamplification reactions as described below. Generally, the amplificationsteps are repeated for a period of time to allow a number of cycles,depending on the number of copies of the original target sequence andthe sensitivity of detection, with cycles ranging from 1 to thousands,with from 10 to 100 cycles being preferred and from 20 to 50 cyclesbeing especially preferred. When linear strand displacementamplification is used cycle numbers can reach thousands to millions.

After a suitable time of amplification, unreacted primers are removed,in a variety of ways, as will be appreciated by those in the art anddescribed below, and the hybridization complex is disassociated. Ingeneral, the modified primer comprises a detectable label, such as afluorescent label, which is either incorporated by the enzyme or presenton the original primer, and the modified primer is detected by any ofthe methods as known to the skilled artisan and include but are notlimited to the methods described herein

Target Amplification

In a preferred embodiment, the amplification is target amplification.Target amplification involves the amplification (replication) of thetarget sequence to be detected, such that the number of copies of thetarget sequence is increased. Suitable target amplification techniquesinclude, but are not limited to, the polymerase chain reaction (PCR),strand displacement amplification (SDA), transcription mediatedamplification (TMA) and nucleic acid sequence based amplification(NASBA).

Polymerase Chain Reaction Amplification

In a preferred embodiment, the target amplification technique is PCR.The polymerase chain reaction (PCR) is widely used and described, andinvolves the use of primer extension combined with thermal cycling toamplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202,and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, allof which are incorporated by reference. In addition, there are a numberof variations of PCR which also find use in the invention, including“quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or“AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformationalpolymorphism” or “PCR-SSCP”, “reverse transcriptase PCR” or “RT-PCR”,“biotin capture PCR”, “vectorette PCR”, “panhandle PCR”, and “PCR selectcDNA subtraction”, “allele-specific PCR”, among others. In someembodiments, PCR is not preferred.

In general, PCR may be briefly described as follows. A double strandedtarget nucleic acid is denatured, generally by raising the temperature,and then cooled in the presence of an excess of a PCR primer, which thenhybridizes to the first target strand. A DNA polymerase then acts toextend the primer with dNTPs, resulting in the synthesis of a new strandforming a hybridization complex. The sample is then heated again, todisassociate the hybridization complex, and the process is repeated. Byusing a second PCR primer for the complementary target strand, rapid andexponential amplification occurs. Thus PCR steps are denaturation,annealing and extension. The particulars of PCR are well known, andinclude the use of a thermostable polymerase such as Taq I polymeraseand thermal cycling.

Accordingly, the PCR reaction requires at least one PCR primer, apolymerase, and a set of dNTPs. As outlined herein, the primers maycomprise the label, or one or more of the dNTPs may comprise a label.

In one embodiment asymmetric PCR is performed. In this embodiment,unequal concentrations of primers are included in the amplificationreaction. The concentrations are designed such that one primer is inexcess or is saturating, while the other primer is limiting or is at asub-saturating concentration.

In one embodiment, PCR primers for amplification of a plurality oftarget nucleic acids are immobilized on a single bead. That is, at leastfirst and second PCR primer pairs are immobilized to a bead ormicrosphere. The microsphere is contacted with a sample and PCRperformed as described herein. Detection of the amplified product orproducts is accomplished by any of the detection methods describedherein, but in a preferred embodiment, detection proceeds byhybridization with allele specific oligonucleotides. That is, uponamplification of the target nucleotides, the immobilized PCR product ishybridized with oligonucleotides that are complementary to the amplifiedproduct.

In a preferred embodiment the allele specific oligonucleotides containdiscrete labels. That is, the oligonucleotides contain distinguishablelabels. As a result of hybridization between the allele specificoligonucleotides and the amplified product(s), detection of a particularlabel provides an indication of the presence of a particular targetnucleic acid in the sample.

In one embodiment, the PCR primers are designed to amplify differentgenomic markers. That is, markers such as translocations or otherchromosomal abnormalities are targeted for amplification. In anadditional embodiment, the primers are designed to amplify genomicregions containing single nucleotide polymorphisms (SNPs). As such, theresulting hybridization with allele specific oligonucleotides providesan indication of the marker or SNP. In one embodiment, a plurality ofmarkers or SNPs is detected on each bead. That is, at least two markersor SNPs are detected on each bead.

In general, as is more fully outlined below, the capture probes on thebeads of the array are designed to be substantially complementary to theextended part of the primer; that is, unextended primers will not bindto the capture probes. Alternatively, as further described below,unreacted probes may be removed prior to addition to the array.

In a preferred embodiment the amplification reaction as a multiplexamplification reaction as described herein. In one embodiment theamplification reaction uses a plurality of PCR primers to amplify aplurality of target sequences. In this embodiment plurality of targetsequences are simultaneously amplified with the plurality ofamplification primer pairs.

An alternative embodiment the multiplex PCR reaction uses universalprimers as described herein. That is, universal PCR primers hybridizedto universal priming sites on the target sequence and thereby amplify aplurality of target sequences. This embodiment is potentially preferredbecause it requires only a limited number of PCR primers. That is, asfew as one primer pairs can amplify a plurality of target sequences.

Strand Displacement Amplification (SDA)

In a preferred embodiment, the target amplification technique is SDA.Strand displacement amplification (SDA) is generally described in Walkeret al., in Molecular Methods for Virus Detection, Academic Press, Inc.,1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which arehereby expressly incorporated by reference in their entirety.

In general, SDA may be described as follows. A single stranded targetnucleic acid, usually a DNA target sequence, is contacted with an SDAprimer. An “SDA primer” generally has a length of 25-100 nucleotides,with SDA primers of approximately 35 nucleotides being preferred. An SDAprimer is substantially complementary to a region at the 3′ end of thetarget sequence, and the primer has a sequence at its 5′ end (outside ofthe region that is complementary to the target) that is a recognitionsequence for a restriction endonuclease, sometimes referred to herein asa “nicking enzyme” or a “nicking endonuclease”, as outlined below. TheSDA primer then hybridizes to the target sequence. The SDA reactionmixture also contains a polymerase (an “SDA polymerase”, as outlinedbelow) and a mixture of all four deoxynucleoside-triphosphates (alsocalled deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), atleast one species of which is a substituted or modified dNTP; thus, theSDA primer is modified, i.e. extended, to form a modified primer,sometimes referred to herein as a “newly synthesized strand”. Thesubstituted dNTP is modified such that it will inhibit cleavage in thestrand containing the substituted dNTP but will not inhibit cleavage onthe other strand. Examples of suitable substituted dNTPs include, butare not limited, 2′deoxyadenosine 5′-O-(1-thiotriphosphate),5-methyldeoxycytidine 5′-triphosphate, 2′-deoxyuridine 5′-triphosphate,and 7-deaza-2′-deoxyguanosine 5′-triphosphate. In addition, thesubstitution of the dNTP may occur after incorporation into a newlysynthesized strand; for example, a methylase may be used to add methylgroups to the synthesized strand. In addition, if all the nucleotidesare substituted, the polymerase may have 5′→3′ exonuclease activity.However, if less than all the nucleotides are substituted, thepolymerase preferably lacks 5′→3′ exonuclease activity.

As will be appreciated by those in the art, the recognitionsite/endonuclease pair can be any of a wide variety of knowncombinations. The endonuclease is chosen to cleave a strand either atthe recognition site, or either 3′ or 5′ to it, without cleaving thecomplementary sequence, either because the enzyme only cleaves onestrand or because of the incorporation of the substituted nucleotides.Suitable recognition site/endonuclease pairs are well known in the art;suitable endonucleases include, but are not limited to, HincII, HindIII,AvaI, Fnu4HI, TthIIII, NclI, BstXI, BamHI, etc. A chart depictingsuitable enzymes, and their corresponding recognition sites and themodified dNTP to use is found in U.S. Pat. No. 5,455,166, herebyexpressly incorporated by reference.

Once nicked, a polymerase (an “SDA polymerase”) is used to extend thenewly nicked strand, 5′→3′, thereby creating another newly synthesizedstrand. The polymerase chosen should be able to initiate 5′→3′polymerization at a nick site, should also displace the polymerizedstrand downstream from the nick, and should lack 5′→3′ exonucleaseactivity (this may be additionally accomplished by the addition of ablocking agent). Thus, suitable polymerases in SDA include, but are notlimited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 andSEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNApolymerase.

Accordingly, the SDA reaction requires, in no particular order, an SDAprimer, an SDA polymerase, a nicking endonuclease, and dNTPs, at leastone species of which is modified. Again, as outlined above for PCR,preferred embodiments utilize capture probes complementary to the newlysynthesized portion of the primer, rather than the primer region, toallow unextended primers to be removed.

In general, SDA does not require thermocycling. The temperature of thereaction is generally set to be high enough to prevent non-specifichybridization but low enough to allow specific hybridization; this isgenerally from about 37° C. to about 42° C., depending on the enzymes.

In a preferred embodiment, as for most of the amplification techniquesdescribed herein, a second amplification reaction can be done using thecomplementary target sequence, resulting in a substantial increase inamplification during a set period of time. That is, a second primernucleic acid is hybridized to a second target sequence, that issubstantially complementary to the first target sequence, to form asecond hybridization complex. The addition of the enzyme, followed bydisassociation of the second hybridization complex, results in thegeneration of a number of newly synthesized second strands.

Nucleic Acid Sequence Based Amplification (NASBA) and TranscriptionMediated Amplification (TMA)

In a preferred embodiment, the target amplification technique is nucleicacid sequence based amplification (NASBA). NASBA is generally describedin U.S. Pat. No. 5,409,818; Sooknanan et al., Nucleic AcidSequence-Based Amplification, Ch. 12 (pp. 261-285) of Molecular Methodsfor Virus Detection, Academic Press, 1995; and “Profiting fromGene-based Diagnostics”, CTB International Publishing Inc., N.J., 1996,all of which are incorporated by reference. NASBA is very similar toboth TMA and QBR. Transcription mediated amplification (TMA) isgenerally described in U.S. Pat. Nos. 5,399,491, 5,888,779, 5,705,365,5,710,029, all of which are incorporated by reference. The maindifference between NASBA and TMA is that NASBA utilizes the addition ofRNAse H to effect RNA degradation, and TMA relies on inherent RNAse Hactivity of the reverse transcriptase.

In general, these techniques may be described as follows. A singlestranded target nucleic acid, usually an RNA target sequence (sometimesreferred to herein as “the first target sequence” or “the firsttemplate”), is contacted with a first primer, generally referred toherein as a “NASBA primer” (although “TMA primer” is also suitable).Starting with a DNA target sequence is described below. These primersgenerally have a length of 25-100 nucleotides, with NASBA primers ofapproximately 50-75 nucleotides being preferred. The first primer ispreferably a DNA primer that has at its 3′ end a sequence that issubstantially complementary to the 3′ end of the first template. Thefirst primer also has an RNA polymerase promoter at its 5′ end (or itscomplement (antisense), depending on the configuration of the system).The first primer is then hybridized to the first template to form afirst hybridization complex. The reaction mixture also includes areverse transcriptase enzyme (an “NASBA reverse transcriptase”) and amixture of the four dNTPs, such that the first NASBA primer is modified,i.e. extended, to form a modified first primer, comprising ahybridization complex of RNA (the first template) and DNA (the newlysynthesized strand).

By “reverse transcriptase” or “RNA-directed DNA polymerase” herein ismeant an enzyme capable of synthesizing DNA from a DNA primer and an RNAtemplate. Suitable RNA-directed DNA polymerases include, but are notlimited to, avian myloblastosis virus reverse transcriptase (“AMV RT”)and the Moloney murine leukemia virus RT. When the amplificationreaction is TMA, the reverse transcriptase enzyme further comprises aRNA degrading activity as outlined below.

In addition to the components listed above, the NASBA reaction alsoincludes an RNA degrading enzyme, also sometimes referred to herein as aribonuclease, that will hydrolyze RNA of an RNA:DNA hybrid withouthydrolyzing single- or double-stranded RNA or DNA. Suitableribonucleases include, but are not limited to, RNase H from E. coli andcalf thymus.

The ribonuclease activity degrades the first RNA template in thehybridization complex, resulting in a disassociation of thehybridization complex leaving a first single stranded newly synthesizedDNA strand, sometimes referred to herein as “the second template”.

In addition, the NASBA reaction also includes a second NASBA primer,generally comprising DNA (although as for all the probes herein,including primers, nucleic acid analogs may also be used). This secondNASBA primer has a sequence at its 3′ end that is substantiallycomplementary to the 3′ end of the second template, and also contains anantisense sequence for a functional promoter and the antisense sequenceof a transcription initiation site. Thus, this primer sequence, whenused as a template for synthesis of the third DNA template, containssufficient information to allow specific and efficient binding of an RNApolymerase and initiation of transcription at the desired site.Preferred embodiments utilizes the antisense promoter and transcriptioninitiation site are that of the T7 RNA polymerase, although other RNApolymerase promoters and initiation sites can be used as well, asoutlined below.

The second primer hybridizes to the second template, and a DNApolymerase, also termed a “DNA-directed DNA polymerase”, also present inthe reaction, synthesizes a third template (a second newly synthesizedDNA strand), resulting in second hybridization complex comprising twonewly synthesized DNA strands.

Finally, the inclusion of an RNA polymerase and the required fourribonucleoside triphosphates (ribonucleotides or NTPs) results in thesynthesis of an RNA strand (a third newly synthesized strand that isessentially the same as the first template). The RNA polymerase,sometimes referred to herein as a “DNA-directed RNA polymerase”,recognizes the promoter and specifically initiates RNA synthesis at theinitiation site. In addition, the RNA polymerase preferably synthesizesseveral copies of RNA per DNA duplex. Preferred RNA polymerases include,but are not limited to, T7 RNA polymerase, and other bacteriophage RNApolymerases including those of phage T3, phage φII, Salmonella phagesp6, or Pseudomonase phage gh-1.

In some embodiments, TMA and NASBA are used with starting DNA targetsequences. In this embodiment, it is necessary to utilize the firstprimer comprising the RNA polymerase promoter and a DNA polymeraseenzyme to generate a double stranded DNA hybrid with the newlysynthesized strand comprising the promoter sequence. The hybrid is thendenatured and the second primer added.

Accordingly, the NASBA reaction requires, in no particular order, afirst NASBA primer, a second NASBA primer comprising an antisensesequence of an RNA polymerase promoter, an RNA polymerase thatrecognizes the promoter, a reverse transcriptase, a DNA polymerase, anRNA degrading enzyme, NTPs and dNTPs, in addition to the detectioncomponents outlined below.

These components result in a single starting RNA template generating asingle DNA duplex; however, since this DNA duplex results in thecreation of multiple RNA strands, which can then be used to initiate thereaction again, amplification proceeds rapidly.

Accordingly, the TMA reaction requires, in no particular order, a firstTMA primer, a second TMA primer comprising an antisense sequence of anRNA polymerase promoter, an RNA polymerase that recognizes the promoter,a reverse transcriptase with RNA degrading activity, a DNA polymerase,NTPs and dNTPs, in addition to the detection components outlined below.

These components result in a single starting RNA template generating asingle DNA duplex; however, since this DNA duplex results in thecreation of multiple RNA strands, which can then be used to initiate thereaction again, amplification proceeds rapidly.

As outlined herein, the detection of the newly synthesized strands canproceed in several ways. Direct detection can be done when the newlysynthesized strands comprise detectable labels, either by incorporationinto the primers or by incorporation of modified labelled nucleotidesinto the growing strand. Alternatively, as is more fully outlined below,indirect detection of unlabelled strands (which now serve as “targets”in the detection mode) can occur using a variety of sandwich assayconfigurations. As will be appreciated by those in the art, any of thenewly synthesized strands can serve as the “target” for form an assaycomplex on a surface with a capture probe. In NASBA and TMA, it ispreferable to utilize the newly formed RNA strands as the target, asthis is where significant amplification occurs.

In this way, a number of secondary target molecules are made. As is morefully outlined below, these reactions (that is, the products of thesereactions) can be detected in a number of ways.

Rolling-Circle Amplification (RCA)

In a preferred embodiment the signal amplification technique is RCA.Rolling-circle amplification is generally described in Baner et al.(1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad.Sci. USA 88:189-193; and Lizardi et al. (1998) Nat. Genet. 19:225-232,all of which are incorporated by reference in their entirety.

In general, RCA may be described in two ways. First, as is outlined inmore detail below, a single probe is hybridized with a target nucleicacid. Each terminus of the probe hybridizes adjacently on the targetnucleic acid and the OLA assay as described above occurs. Alternatively,two probes are hybridized with the target nucleic acid and the OLA assayas described above occurs. When ligated, the probe is circularized whilehybridized to the target nucleic acid, or a circular primer is added tothe ligated target nucleic acid complex. Addition of a polymeraseresults in extension of the circular probe. However, since the probe hasno terminus, the polymerase continues to extend the probe repeatedly.Thus results in amplification of the circular probe.

A second alternative approach involves OLA followed by RCA. In thisembodiment, an immobilized primer is contacted with a target nucleicacid. Complementary sequences will hybridize with each other resultingin an immobilized duplex. A second primer is contacted with the targetnucleic acid. The second primer hybridizes to the target nucleic acidadjacent to the first primer. An OLA assay is performed as describedabove. Ligation only occurs if the primer are complementary to thetarget nucleic acid. When a mismatch occurs, particularly at one of thenucleotides to be ligated, ligation will not occur. Following ligationof the oligonucleotides, the ligated, immobilized, oligonucleotide isthen hybridized with an RCA probe. This is a circular probe that isdesigned to specifically hybridize with the ligated oligonucleotide andwill only hybridize with an oligonucleotide that has undergone ligation.RCA is then performed as is outlined in more detail below.

Accordingly, in an preferred embodiment, a single oligonucleotide isused both for OLA and as the circular template for RCA (referred toherein as a “padlock probe” or a “RCA probe”). That is, each terminus ofthe oligonucleotide contains sequence complementary to the targetnucleic acid and functions as an OLA primer as described above. That is,the first end of the RCA probe is substantially complementary to a firsttarget domain, and the second end of the RCA probe is substantiallycomplementary to a second target domain, adjacent to the first domain.Hybridization of the oligonucleotide to the target nucleic acid resultsin the formation of a hybridization complex. Ligation of the “primers”(which are the discrete ends of a single oligonucleotide) results in theformation of a modified hybridization complex containing a circularprobe i.e. an RCA template complex. That is, the oligonucleotide iscircularized while still hybridized with the target nucleic acid. Thisserves as a circular template for RCA. Addition of a polymerase to theRCA template complex results in the formation of an amplified productnucleic acid. Following RCA, the amplified product nucleic acid isdetected. This can be accomplished in a variety of ways; for example,the polymerase may incorporate labeled nucleotides, or alternatively, alabel probe is used that is substantially complementary to a portion ofthe RCA probe and comprises at least one label is used.

The polymerase can be any polymerase, but is preferably one lacking 3′exonuclease activity (3′ exo⁻). Examples of suitable polymerase includebut are not limited to exonuclease minus DNA Polymerase I large (Klenow)Fragment, Phi29 DNA polymerase, Taq DNA Polymerase and the like. Inaddition, in some embodiments, a polymerase that will replicatesingle-stranded DNA (i.e. without a primer forming a double strandedsection) can be used.

In a preferred embodiment, the RCA probe contains an adapter sequence asoutlined herein, with adapter capture probes on the array, for exampleon a microsphere when microsphere arrays are being used. Alternatively,unique portions of the RCA probes, for example all or part of thesequence corresponding to the target sequence, can be used to bind to acapture probe.

In a preferred embodiment, the padlock probe contains a restrictionsite. The restriction endonuclease site allows for cleavage of the longconcatamers that are typically the result of RCA into smaller individualunits that hybridize either more efficiently or faster to surface boundcapture probes. Thus, following RCA, the product nucleic acid iscontacted with the appropriate restriction endonuclease. This results incleavage of the product nucleic acid into smaller fragments. Thefragments are then hybridized with the capture probe that is immobilizedresulting in a concentration of product fragments onto the microsphere.Again, as outlined herein, these fragments can be detected in one of twoways: either labelled nucleotides are incorporated during thereplication step, or an additional label probe is added.

Thus, in a preferred embodiment, the padlock probe comprises a labelsequence; i.e. a sequence that can be used to bind label probes and issubstantially complementary to a label probe. In one embodiment, it ispossible to use the same label sequence and label probe for all padlockprobes on an array; alternatively, each padlock probe can have adifferent label sequence.

The padlock probe also contains a priming site for priming the RCAreaction. That is, each padlock probe comprises a sequence to which aprimer nucleic acid hybridizes forming a template for the polymerase.The primer can be found in any portion of the circular probe. In apreferred embodiment, the primer is located at a discrete site in theprobe. In this embodiment, the primer site in each distinct padlockprobe is identical, although this is not required. Advantages of usingprimer sites with identical sequences include the ability to use only asingle primer oligonucleotide to prime the RCA assay with a plurality ofdifferent hybridization complexes. That is, the padlock probe hybridizesuniquely to the target nucleic acid to which it is designed. A singleprimer hybridizes to all of the unique hybridization complexes forming apriming site for the polymerase. RCA then proceeds from an identicallocus within each unique padlock probe of the hybridization complexes.

In an alternative embodiment, the primer site can overlap, encompass, orreside within any of the above-described elements of the padlock probe.That is, the primer can be found, for example, overlapping or within therestriction site or the identifier sequence. In this embodiment, it isnecessary that the primer nucleic acid is designed to base pair with thechosen primer site.

Thus, the padlock probe of the invention contains at each terminus,sequences corresponding to OLA primers. The intervening sequence of thepadlock probe contain in no particular order, an adapter sequence and arestriction endonuclease site. In addition, the padlock probe contains aRCA priming site.

Thus, in a preferred embodiment the OLA/RCA is performed in solutionfollowed by restriction endonuclease cleavage of the RCA product. Thecleaved product is then applied to an array comprising beads, each beadcomprising a probe complementary to the adapter sequence located in thepadlock probe. The amplified adapter sequence correlates with aparticular target nucleic acid. Thus the incorporation of anendonuclease site allows the generation of short, easily hybridizablesequences. Furthermore, the unique adapter sequence in each rollingcircle padlock probe sequence allows diverse sets of nucleic acidsequences to be analyzed in parallel on an array, since each sequence isresolved on the basis of hybridization specificity.

In an alternative OLA/RCA method, one of the OLA primers is immobilizedon the microsphere; the second primer is added in solution. Both primershybridize with the target nucleic acid forming a hybridization complexas described above for the OLA assay.

As described herein, the microsphere is distributed on an array. In apreferred embodiment, a plurality of microspheres each with a unique OLAprimer is distributed on the array.

Following the OLA assay, and either before, after or concurrently withdistribution of the beads on the array, a segment of circular DNA ishybridized to the bead-based ligated oligonucleotide forming a modifiedhybridization complex. Addition of an appropriate polymerase (3′ exo⁻),as is known in the art, and corresponding reaction buffer to the arrayleads to amplification of the circular DNA. Since there is no terminusto the circular DNA, the polymerase continues to travel around thecircular template generating extension product until it detaches fromthe template. Thus, a polymerase with high processivity can createseveral hundred or thousand copies of the circular template with all thecopies linked in one contiguous strand.

Again, these copies are subsequently detected by one of two methods;either hybridizing a labeled oligo complementary to the circular targetor via the incorporation of labeled nucleotides in the amplificationreaction. The label is detected using conventional label detectionmethods as described herein.

In one embodiment, when the circular DNA contains sequencescomplementary to the ligated oligonucleotide it is preferable to removethe target DNA prior to contacting the ligated oligonucleotide with thecircular DNA (See FIG. 7). This is done by denaturing thedouble-stranded DNA by methods known in the art. In an alternativeembodiment, the double stranded DNA is not denatured prior to contactingthe circular DNA.

In an alternative embodiment, when the circular DNA contains sequencescomplementary to the target nucleic acid, it is preferable that thecircular DNA is complementary at a site distinct from the site bound tothe ligated oligonucleotide. In this embodiment it is preferred that theduplex between the ligated oligonucleotide and target nucleic acid isnot denatured or disrupted prior to the addition of the circular DNA sothat the target DNA remains immobilized to the bead.

Hybridization and washing conditions are well known in the art; variousdegrees of stringency can be used. In some embodiments it is notnecessary to use stringent hybridization or washing conditions as onlymicrospheres containing the ligated probes will effectively hybridizewith the circular DNA; microspheres bound to DNA that did not undergoligation (those without the appropriate target nucleic acid) will nothybridize as strongly with the circular DNA as those primers that wereligated. Thus, hybridization and/or washing conditions are used thatdiscriminate between binding of the circular DNA to the ligated primerand the unligated primer.

Alternatively, when the circular probe is designed to hybridize to thetarget nucleic acid at a site distinct from the site bound to theligated oligonucleotide, hybridization and washing conditions are usedto remove or dissociate the target nucleic acid from unligatedoligonucleotides while target nucleic acid hybridizing with the ligatedoligonucleotides will remain bound to the beads. In this embodiment, thecircular probe only hybridizes to the target nucleic acid when thetarget nucleic acid is hybridized with a ligated oligonucleotide that isimmobilized on a bead.

As is well known in the art, an appropriate polymerase (3′ exo⁻) isadded to the array. The polymerase extends the sequence of asingle-stranded DNA using double-stranded DNA as a primer site. In oneembodiment, the circular DNA that has hybridized with the appropriateOLA reaction product serves as the primer for the polymerase. In thepresence of an appropriate reaction buffer as is known in the art, thepolymerase will extend the sequence of the primer using thesingle-stranded circular DNA as a template. As there is no terminus ofthe circular DNA, the polymerase will continue to extend the sequence ofthe circular DNA. In an alternative embodiment, the RCA probe comprisesa discrete primer site located within the circular probe. Hybridizationof primer nucleic acids to this primer site forms the polymerasetemplate allowing RCA to proceed.

In a preferred embodiment, the polymerase creates more than 100 copiesof the circular DNA. In more preferred embodiments the polymerasecreates more than 1000 copies of the circular DNA; while in a mostpreferred embodiment the polymerase creates more than 10,000 copies ormore than 50,000 copies of the template.

The amplified circular DNA sequence is then detected by methods known inthe art and as described herein. Detection is accomplished byhybridizing with a labeled probe. The probe is labeled directly orindirectly. Alternatively, labeled nucleotides are incorporated into theamplified circular DNA product. The nucleotides can be labeled directly,or indirectly as is further described herein.

The RCA as described herein finds use in allowing highly specific andhighly sensitive detection of nucleic acid target sequences. Inparticular, the method finds use in improving the multiplexing abilityof DNA arrays and eliminating costly sample or target preparation. As anexample, a substantial savings in cost can be realized by directlyanalyzing genomic DNA on an array, rather than employing an intermediatePCR amplification step. The method finds use in examining genomic DNAand other samples including mRNA.

In addition the RCA finds use in allowing rolling circle amplificationproducts to be easily detected by hybridization to probes in asolid-phase format (e.g. an array of beads). An additional advantage ofthe RCA is that it provides the capability of multiplex analysis so thatlarge numbers of sequences can be analyzed in parallel. By combining thesensitivity of RCA and parallel detection on arrays, many sequences canbe analyzed directly from genomic DNA.

In an alternative embodiment, the OLA assay includes employing astandard solution phase OLA assay using adapter sequences to capture theOLA product. In this case, the allele specific oligonucleotides alsocontain a sequence that is complementary to a circular RCA primer thatis indicative of the respective allele. That is, the OLA primer designedto hybridize to one allele contains a specific sequence forhybridization to a specific RCA primer. Likewise, the OLA primerdesigned to hybridize to the second allele contains a specific sequencefor hybridization to a second specific RCA primer. Following OLA andcapture of the OLA product, both RCA primers are hybridized with the OLAproduct, but only the RCA primer that is complementary to the respectiveRCA primer site will hybridize with that site. An RCA assay is performedand the product detected as described herein. The RCA product is anindication of the presence of a particular allele.

In one embodiment RCA is used to amplify cDNA. As is known in the art,cDNA is obtained by reverse transcription of mRNA. The resulting cDNA,therefore is a representation of the mRNA population in a given sample.Accordingly, it is desirable to examine cDNA to gain insight into therelative level of mRNA of a sample. However, frequently there exists aneed to amplify the cDNA in order to obtain sufficient quantities forvarious analyses. Previously, amplification strategies involvedexponential techniques such as PCR. A potential problem with exponentialamplification is that it occasionally results in distorted mRNAprofiles. Given the desire to examine mRNA populations, which provide anindication of the expression level of different gene products, there isa desire to develop amplification techniques that provide a moreaccurate indication of the mRNA levels in a sample.

Accordingly, the present invention provides a method of amplifying cDNAusing the RCA as described herein. In a preferred embodiment, the methodincludes circularizing the cDNA and amplifying the circularizedsubstrate with a DNA polymerase. In a preferred embodiment the cDNA iscircularized by hybridization with a “guide linker”. By “guide linker”is meant an oligonucleotide that is complementary to the 5′ and 3′termini of the cDNA molecule. Generally, the 5′ terminus of a cDNAmolecule contains a poly-T track. In addition, the 3′ terminus of cDNAfrequently contains multiple C nucleotides. Generally three or four Cnucleotides are added to the 3′ terminus of the cDNA. Without beingbound by theory, it is thought that these Cs are a result ofnon-template mediated addition of the C nucleotides to the 3′ terminusby the DNA Polymerase. Accordingly, in a preferred embodiment the guidelinker contains a plurality of A nucleotides at one terminus and aplurality of G nucleotides at the other terminus. That is, it containsat its 5′ terminus a plurality of G nucleotides and at its 3′ terminus aplurality of A nucleotides. A preferred guide linker contains thesequence GGGAAAA, although it could contain more or fewer Gs or As ateach of the respective termini.

Upon hybridization of the guide linker with the cDNA, the circular cDNAis covalently closed following incubation with ligase. That is,incubation with ligase results in covalent attachment of the 5′T and 3°C. of the cDNA). The circular cDNA/guide linker complex is thencontacted with a DNA polymerase that extends the circular template asdescribed herein. The cDNA/guide linker complex serves as a template forthe polymerase. This results in linear amplification of the cDNA andresults in a population of cDNA that is representative of the mRNAlevels of a sample. That is, the amplified cDNA provides an indicationof the gene expression level of a sample. In addition, the amplifiedproducts represent full length cDNAs as a result of selection with aguide linker that contains a poly-T tract and a poly-G tract.

As described herein, in some embodiments labeled nucleotides areincorporated into the amplified cDNA product. This results in linearamplification of the signal.

The amplified cDNA product finds use in a variety of assays includinggene expression analysis. The amplified products find use as probes thatcan be applied to an array as described herein.

Cycling Probe Technology (CPT Cycling probe technology (CPT) is anucleic acid detection system based on signal or probe amplificationrather than target amplification, such as is done in polymerase chainreactions (PCR). Cycling probe technology relies on a molar excess oflabeled probe which contains a scissile linkage of RNA. Uponhybridization of the probe to the target, the resulting hybrid containsa portion of RNA:DNA. This area of RNA:DNA duplex is recognized byRNAseH and the RNA is excised, resulting in cleavage of the probe. Theprobe now consists of two smaller sequences which may be released, thusleaving the target intact for repeated rounds of the reaction. Theunreacted probe is removed and the label is then detected. CPT isgenerally described in U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988,and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416,and WO 95/00667, all of which are specifically incorporated herein byreference.

Oligonucleotide Ligation Assay

The oligonucleotide ligation assay (OLA; sometimes referred to as theligation chain reaction (LCR)) involve the ligation of at least twosmaller probes into a single long probe, using the target sequence asthe template for the ligase. See generally U.S. Pat. Nos. 5,185,243,5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182B1; WO 90/01069; WO 89/12696; and WO 89/09835, all of which areincorporated by reference.

Invader™

Invader™ technology is based on structure-specific polymerases thatcleave nucleic acids in a site-specific manner. Two probes are used: an“invader” probe and a “signaling” probe, that adjacently hybridize to atarget sequence with a non-complementary overlap. The enzyme cleaves atthe overlap due to its recognition of the “tail”, and releases the“tail” with a label. This can then be detected. The Invader™ technologyis described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028;5,541,311; and 5,843,669, all of which are hereby incorporated byreference.

ICAN Amplification

ICAN methodology is a preferred amplification method that includeshybridizing chimeric-primers composed of RNA (3′ end) and DNA (5′ end)and providing a DNA polymerase with strand displacement activity(BcaBEST™ DNA polymerase from Takara Shuzo Co., Ltd), which extends theprimer forming a double stranded intermediate. Subsequently, aribonuclease cleaves the junction of the DNA-RNA hybrid (RNase H).Subsequently, an additional chimeric primer hybridizes with theextension product or original target and displaces one strand of thedouble stranded intermediate. This cycle is repeated thereby amplifyingthe target. Amplification is outlined in FIG. 12. In a preferredembodiment ICAN method can be used to amplify specific regions of DNA ata constant temperature of 50 to 65° C. That is, the amplification isisothermal.

SPIA™

In a preferred embodiment, a linear amplification scheme known as ESPIA,or SPIA is applied. This amplification technique is disclosed in WO01/20035 A2 and U.S. Pat. No. 6,251,639, which are incorporated byreference herein. Generally, the method includes hybridizing chimericRNA/DNA amplification primers to the probes or target. Preferably theDNA portion of the probe is 3′ to the RNA. Optionally the methodincludes hybridizing a polynucleotide comprising a terminationpolynucleotide sequence to a region of the template that is 5′ withrespect to hybridization of the composite primer to the template.Following hybridization of the primer to the template, the primer isextended with DNA polymerase. Subsequently, the RNA is cleaved from thecomposite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid.Subsequently, an additional RNA/DNA chimeric primer is hybridized to thetemplate such that the first extended primer is displaced from thetarget probe. The extension reaction is repeated, whereby multiplecopies of the probe sequence are generated.

Amplicon Enrichment

In this alternate method, following amplification, as described above,the amplicons are hybridized to a solid-phase containing immobilizedtargets, i.e. genomic DNA or oligonucleotides corresponding to targetedSNPs. Preferably the amplification primers include universal primers, asdescribed herein. In a preferred embodiment hybridization is performedat high temperatures such that only the desired PCR products (those thatinclude or span the particular allele) are retained, while non-specificproducts or primer-dimers, which have a reduced Tm are removed bywashing. That is, the notable difference between the Tms of specificproducts, which are preferably form 65 to 85° C., more preferably form70 to 80° C., and the Tms of the non-specific products, which is aroundfrom about 45-60° C., provides a separation window for controlling ordiscriminating between the two populations during hybridization andwashing.

The immobilized target can be any nucleic acid as described herein.Preferably the immobilized target is genomic DNA or oligonucleotidescorresponding to particular SNPs. Alternatively, it could be pooledgenomic DNA from a variety of sources or individually amplifiedproducts.

Once the non-specific products have been removed, the retained PCRproducts may be detected. Alternatively, they may be additionallyamplified. Alternatively, they may be used in any genotyping assays asare known in the art and described herein.

Label

By “detection label” or “detectable label” herein is meant a moiety thatallows detection. This may be a primary label or a secondary label.Accordingly, detection labels may be primary labels (i.e. directlydetectable) or secondary labels (indirectly detectable).

In a preferred embodiment, the detection label is a primary label. Aprimary label is one that can be directly detected, such as afluorophore. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal labels; and c) colored or luminescent dyes. Labelscan also include enzymes (horseradish peroxidase, etc.) and magneticparticles. Preferred labels include chromophores or phosphors but arepreferably fluorescent dyes. Suitable dyes for use in the inventioninclude, but are not limited to, fluorescent lanthanide complexes,including those of Europium and Terbium, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,quantum dots (also referred to as “nanocrystals”: see U.S. Ser. No.09/315,584, hereby incorporated by reference), pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, Cy dyes (Cy3, Cy5,etc.), alexa dyes, phycoerythin, bodipy, and others described in the 6thEdition of the Molecular Probes Handbook by Richard P. Haugland, herebyexpressly incorporated by reference.

In a preferred embodiment, a secondary detectable label is used. Asecondary label is one that is indirectly detected; for example, asecondary label can bind or react with a primary label for detection,can act on an additional product to generate a primary label (e.g.enzymes), or may allow the separation of the compound comprising thesecondary label from unlabeled materials, etc. Secondary labels findparticular use in systems requiring separation of labeled and unlabeledprobes, such as SBE, OLA, invasive cleavage reactions, etc; in addition,these techniques may be used with many of the other techniques describedherein. Secondary labels include, but are not limited to, one of abinding partner pair; chemically modifiable moieties; nucleaseinhibitors, enzymes such as horseradish peroxidase, alkalinephosphatases, lucifierases, etc.

In a preferred embodiment, the secondary label is a binding partnerpair. For example, the label may be a hapten or antigen, which will bindits binding partner. In a preferred embodiment, the binding partner canbe attached to a solid support to allow separation of extended andnon-extended primers. For example, suitable binding partner pairsinclude, but are not limited to: antigens (such as proteins (includingpeptides)) and antibodies (including fragments thereof (FAbs, etc.));proteins and small molecules, including biotin/streptavidin; enzymes andsubstrates or inhibitors; other protein-protein interacting pairs;receptor-ligands; and carbohydrates and their binding partners. Nucleicacid-nucleic acid binding proteins pairs are also useful. In general,the smaller of the pair is attached to the NTP for incorporation intothe primer. Preferred binding partner pairs include, but are not limitedto, biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, andProlinx™ reagents (see www.prolinxinc.com/ie4/home.hmtl).

In a preferred embodiment, the binding partner pair comprises biotin orimino-biotin and streptavidin. Imino-biotin is particularly preferred asimino-biotin disassociates from streptavidin in pH 4.0 buffer whilebiotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or90% formamide at 95° C.).

In a preferred embodiment, the binding partner pair comprises a primarydetection label (for example, attached to the NTP and therefore to theextended primer) and an antibody that will specifically bind to theprimary detection label. By “specifically bind” herein is meant that thepartners bind with specificity sufficient to differentiate between thepair and other components or contaminants of the system. The bindingshould be sufficient to remain bound under the conditions of the assay,including wash steps to remove non-specific binding. In someembodiments, the dissociation constants of the pair will be less thanabout 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ beingpreferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ being particularlypreferred.

In a preferred embodiment, the secondary label is a chemicallymodifiable moiety. In this embodiment, labels comprising reactivefunctional groups are incorporated into the nucleic acid. The functionalgroup can then be subsequently labeled with a primary label. Suitablefunctional groups include, but are not limited to, amino groups, carboxygroups, maleimide groups, oxo groups and thiol groups, with amino groupsand thiol groups being particularly preferred. For example, primarylabels containing amino groups can be attached to secondary labelscomprising amino groups, for example using linkers as are known in theart; for example, homo- or hetero-bifunctional linkers as are well known(see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).

However, in this embodiment, the label is a secondary label, apurification tag, that can be used to capture the sequence comprisingthe tag onto a second solid support surface.

The addition of the polymerase and the labeled dNTP are done underconditions to allow the formation of a modified first probe. Themodified first probe is then added to a second solid support using thepurification tag as outlined herein.

Once immobilized, several reagents are adding to the modified probe. Ina preferred embodiment, first and second universal probes are added,with a polymerase and dNTPs, such that the modified probe is amplifiedto form amplicons, which can then be detected on arrays as outlinedbelow. While the figures are generally directed to PCR systems, otheramplification systems can be used, as are generally outlined in Ser. No.09/517,945, filed Mar. 3, 2000, 60/161,148, filed Oct. 22, 1999,60/135,051, filed May 20, 1999, 60/244,119, filed Oct. 26, 2000, Ser.No. 09/556,463, filed Apr. 21, 2000, and Ser. No. 09/553,993, filed Apr.20, 2000, all of which are expressly incorporated herein by reference.

Combination Techniques

Other preferred configurations of the system are shown in the figures.

In one embodiment the target nucleic acid is first immobilized. This isfollowed by a specificity step, i.e. allele specific extension (seeFIG. 1) and amplification. That is, following immobilization of thetarget nucleic acids, the target nucleic acids are contacted with allelespecific probes under stringent annealing conditions. Non-hybridizedprobes are removed by a stringent wash. Subsequently the hybridizedprobes or primers are contacted with an enzyme such as a polymerase inthe presence of labeled ddNTP (see FIG. 1) forming a modified primer.Preferably the label is a purification tag as described herein. TheddNTP is only incorporated into the primer that is perfectlycomplementary to the target nucleic acid. The modified primer is theneluted from the immobilized target nucleic acid, and contacted withamplification primers to form amplicons. In one embodiment the elutedprimer is purified by binding to a binding partner for the affinity tag.Then the purified and modified primer is contacted with amplificationprimers for amplification, forming amplicons. The amplicons are thendetected as an indication of the presence of the particular targetnucleic acid.

In a preferred embodiment, as shown in FIG. 1, the allele specificprimer also includes an adapter sequence and priming sequences. That is,the primer includes from 5′ to 3′, and upstream amplification primingsite, an adapter sequence, a downstream amplification priming site, andan allele specific sequence Priming sequences hybridize withamplification primers; the adapter sequence mediates attachment of theamplicons to a support for subsequent detection of amplicons. Inpreferred embodiments, as described herein, the priming sequences areuniversal priming sequences. This allows for highly multiplexedamplification. In a preferred embodiment at least one of the universalpriming sequences is specific for a particular allele.

As shown in the figures, allele detection can proceed on a number oflevels. In one embodiment adapters are distinct for the particularallele. Thus, following amplification of the adapter sequences,detection of the adapter provides identification of the particularallele to be detected.

Alternatively, allele detection proceeds as a result of allele specificamplification. As shown in FIG. 1, at least one of the priming sequenceson the primers for each allele is specific for a particular allele.Thus, following the specificity assay, one of the alleles will beidentified. Following addition of the respective amplification primers,only one set of the primers will hybridize with the priming sequences.Thus, only one of the sets of primers will generate an amplicon. In apreferred embodiment, each of the sets of primers is labeled withdistinct label. Because only one of the sets will be amplified,detection of a label provides an indication of the primer that wasamplified. This, in turn identifies the nucleotide at the detectionposition.

In an alternative embodiment the target nucleic acid is first contactedwith a first target specific probe under stringent annealing conditionsand a first extension reaction is performed with either dNTPs or ddNTPSforming a first extension product (see FIG. 2). The first targetspecific probe in this embodiment is either a locus specific probe or anallele specific probe. This step reduces the complexity of the sample.Subsequently the first extension product is contacted with a secondprobe that has the same sequence as a portion of the target sequence,i.e. the second probe is complementary to the extension product, andagain can be either an allele specific probe or a locus specific probe.Following hybridization of the second probe, a second extension reactionis performed.

In a preferred embodiment the primers for the first and second extensionreaction also include amplification priming sites. Preferably theamplification priming sites are universal priming sites as describedherein. Accordingly, the resulting extension product is amplified (theamplification component of the multiplexing scheme). The resultingdouble stranded product is then denatured and either of the strands isused as a template for a single base extension (SBE) reaction asdescribed in more detail below (the specificity component). In the SBEreaction, chain terminating nucleotides such as ddTNPs are used assubstrates for the polymerase and are incorporated into a target probethat is hybridized to the single stranded amplicon template adjacent tothe interrogation position. Preferably the ddNTPs are labeled asdescribed below. Preferably, the ddNTPs are discretely labeled such thatthey can be discriminated in the detection step.

In an alternative embodiment a first biotinylated or otherwise taggedprobe is hybridized with a target nucleic acid and a first extensionreaction is performed. The primer or probe is either an allele specificor locus specific probe. The extended product is then purified from themixture by the tag. Again, this serves as the complexity reduction step.Subsequently, a second primer is hybridized to the first extensionproduct and a second extension reaction is performed, preferably in anallele specific manner, i.e. with discriminatory probes that arespecific for each allele. This represents the specificity step.Preferably, both of the primers used in the extension reactions containuniversal priming sites. Thus, universal primers can be added foruniversal amplification of the extension products (the amplificationcomponent) (see FIG. 3). In a preferred embodiment, each allele specificprimer includes a distinct amplification priming site. Thus, followingallele discrimination, only one of the primers can be used foramplification, resulting in allele specific amplification. Preferablythe amplification primers contain discrete labels, which again allowsfor detection of which particular primers served as amplificationtemplates. This, again, identifies the particular allele to be detected.In an additional preferred embodiment, at least one of the primersincludes an adapter sequence as outlined below.

In an alternative embodiment tagged, i.e. biotinylated, primers arehybridized with a target nucleic acid. Preferably the hybridizationcomplex is immobilized. Either the target or the primer can be theimmobilized component. After annealing, the immobilized complexes arewashed to remove unbound nucleic acids. This is followed by an extensionreaction. This is the complexity reduction component of the assay.Subsequently, the extended probe is removed via the purification tag.The purified probe is then hybridized with allele specific probes (thespecificity component). The hybridized probes are then amplified (theamplification component) (see FIG. 4).

In a preferred embodiment the allele specific probe contains universalpriming sites and an adapter sequence. Preferably the universal primingsites are specific for a particular allele. That is, one of theuniversal priming sites may be common to all alleles, but the seconduniversal priming site is specific for a particular allele. Followinghybridization the allele specific primer, the complexes are washed toremove unbound or mismatched primers. Thus, this configuration allowsfor allele specific amplification. Amplicons are detected as anindication of the presence of a particular allele.

In an alternative embodiment, the specificity component occurs first, Inthis embodiment allele specific probes are hybridized with the targetnucleic acid; an extension assay is performed whereby only the perfectlycomplementary probe is extended. That is, only the probe that isperfectly complementary to the probe at the interrogation positionserves as a substrate for extension reaction. Preferably the extensionreaction includes tagged, i.e. biotinylated, dNTPs such that theextension product is tagged. The extension product is then purified fromthe reaction mixture. Subsequently, a second allele specific primer ishybridized to the extension product. This step also serves as a secondspecificity step. In this embodiment the specificity steps also serve ascomplexity reduction components in that they enrich for target nucleicacids. Following the addition of the second allele specific primer andextension, the extension product is amplified, preferably with universalprimers (see FIG. 5).

as discussed previously, it is preferably for the at least one allelespecific primer to contain an allele specific priming site, preferablyan allele specific universal priming site. Again, this configurationallows for multiplexed allele specific amplification using universalprimers.

In an alternative embodiment, the target nucleic acid is firstimmobilized and hybridized with allele specific primers. Preferably theallele specific primers also include an adapter sequence that isindicative of the particular allele. Allele specific extension is thenperformed whereby only the primer that is perfectly complementary to thedetection position of the target nucleic acid will serve as a templatefor primer extension. That is, mismatched primers will not be extended.Of note, the allele specific position of the primer need not be the 3′terminal nucleotide of the primer (see FIGS. 7 and 8). That is, theprimer may extend beyond the detection position of the target nucleicacid. In this embodiment it is preferable to include labeled dNTPs orddNTPs or both such that the extension product is labeled and can bedetected. In some preferred embodiments the interrogator is not theterminal position of the primer, but rather resides at a position 1, 2,3, 4, 5 or 6 nucleotides from the 3′ terminus of the primer.

In a preferred embodiment both dNTPs and ddNTPs are included in theextension reaction mixture. In this embodiment only one label is needed,and the amount of label can be determined and altered by varying therelative concentration of labeled and unlabeled dTNPs and ddNTPs. Thatis, in one embodiment labeled ddNTPs are included in the extension mixat a dilution such that each termination will result in placement ofsingle label on each strand. Thus, this method allows for quantificationof targets. Alternatively, if a higher signal is needed, a mixture oflabeled dNTPs can be used along with chain terminating nucleotides at alower concentration. The result is the incorporation of multiple labelsper extension product. Preferably the primers also include adapterswhich facilitate immobilization of the extension products for detection.

In an additional preferred configuration, target nucleic acids arehybridized with tagged locus specific primers. Preferably the primerincludes a locus specific portion and a universal priming site. Of note,as is generally true for locus specific primers, they need not beimmediately adjacent to the detection position. Upon hybridization, thehybridization complexes are immobilized, preferably by binding moietythat specifically binds the tag on the locus specific primer. Theimmobilized complexes are then washed to remove unlabeled nucleic acids;the remaining hybridization complexes are then subject to an extensionreaction. Following extension of the locus specific primer, a nucleotidecomplementary to the nucleotide at the detection position will beincorporated into the extension product. In some embodiments it isdesirable to limit the size of the extension because this reduces thecomplexity of subsequent annealing steps. This may be accomplished byincluding both dNTPs and ddNTPs in the reaction mixture.

Following the first extension, a second locus or allele specific primeris hybridized to the immobilized extension product and a secondextension reaction occurs. Preferably the second extension primerincludes a target specific portion and a universal priming site. Afterextension, universal amplification primers can be added to the reactionand the extension products amplified. The amplicons can then be used fordetection of the particular allele. This can be accomplished bycompetitive hybridization, as described herein. Alternatively, it can beaccomplished by an additional extension reaction. When the extensionreaction is performed, preferably a primer that contains an adaptersequence and a target specific portion is hybridized with the amplicons.Preferably the target specific portion hybridizes up to a position thatis adjacent to the detection position, i.e. the particular allele to bedetected. Polymerase and labeled ddNTPs are then added and the extensionreaction proceeds, whereby incorporation of a particular label isindicative of the nucleotide that is incorporated into the extensionprimer. This nucleotide is complementary to the nucleotide at thedetection position. Thus, analyzing or detecting which nucleotide isincorporated into the primer provides an indication of the nucleotide atthe allele position. The extended primer is detected by methods thatinclude but are not limited to the methods described herein.

In another embodiment, the genotyping specificity is conferred by theextension reaction. In this embodiment, two probes (sometimes referredto herein as “primers”) are hybridized non-contiguously to a targetsequence comprising, from 3′ to 5′, a first second and third targetdomain. Preferably the target is immobilized. That is, in a preferredembodiment, the target sequence is genomic DNA and is attached to asolid support as is generally described in U.S. Ser. No. 09/931,285,hereby expressly incorporated by reference in its entirety. In thisembodiment, magnetic beads, tubes or microtiter plates are particularlypreferred solid supports, although other solid supports as describedbelow can also be used.

The first probe hybridized to the first domain, contains a firstuniversal priming sequence and contains, at the 3′ end (within theterminal six bases), an interrogation position. In some preferredembodiments the interrogator is not the terminal position of the primer,but rather resides at a position 1, 2, 3, 4, 5 or 6 nucleotides from the3′ terminus of the primer. Subsequently, the unhybridized primers areremoved. This is followed by providing an extension enzyme such as apolymerase, and NTPs (which includes both dNTPs, NTPs and analogs, asoutlined below). If the interrogation position is perfectlycomplementary to the detection position of the target sequence, theextension enzyme will extend through the second target domain to form anextended first probe, ending at the beginning of the third domain, towhich the second probe is hybridized. A second probe is complementary tothe third target domain, and upon addition of a ligase, the extendedfirst probe will ligate to the second probe. The addition of a primerallows amplification to form amplicons. If the second probe comprises anantisense second primer, exponential amplification may occur, such as inPCR. Similarly, one or other of the probes may comprise an adapter oraddress sequence, which facilitates detection. For example, the adaptermay serve to allow hybridization to a “universal array”. Alternatively,the adapter may serve as a mobility modifier for electrophoresis or massspectrometry analysis, or as a label sequence for the attachment oflabels or beads for flow cytometry analysis.

In another embodiment, the reaction is similar except that it is theligation reaction that provides the detection position/interrogationspecificity. In this embodiment, it is the second probe that comprises a5′ interrogation position. The extended first probe will not be ligatedto the second probe if there is a mismatch between the interrogationposition and the target sequence. As above, the addition of a primerallows amplification to form amplicons. If the second probe comprises anantisense second primer, exponential amplification may occur, such as inPCR. Similarly, one or other of the probes may comprise an adapter oraddress sequence, which facilitates detection. For example, the adaptermay serve to allow hybridization to a “universal array”. Alternatively,the adapter may serve as a mobility modifier for electrophoresis or massspectrometry analysis, or as a label sequence for the attachment oflabels or beads for flow cytometry analysis.

Once prepared, and attached to a solid support as required, the targetsequence is used in genotyping reactions. It should be noted that whilethe discussion below focuses on certain assays, in general, for eachreaction, each of these techniques may be used in a solution basedassay, wherein the reaction is done in solution and a reaction productis bound to the array for subsequent detection, or in solid phaseassays, where the reaction occurs on the surface and is detected, eitheron the same surface or a different one.

The assay continues with the addition of a first probe. The first probecomprises, a 5′ first domain comprising a first universal primingsequence. The universal priming sites are used to amplify the modifiedprobes to form a plurality of amplicons that are then detected in avariety of ways, as outlined herein. In preferred embodiments, one ofthe universal priming sites is a T7 site, such that RNA is ultimatelymade to form the amplicon. Alternatively, as more fully outlined below,two universal priming sequences are used, one on the second probegenerally in antisense orientation, such that PCR reactions or otherexponential amplification reactions can be done. Alternatively, a singleuniversal primer can be used for amplification. Linear amplification canbe performed using the SPIA assay, T7 amplification, linear TMA and thelike, as described herein.

The first probe further comprises, 3′ to the priming sequence, a seconddomain comprising a sequence substantially complementary to the firsttarget domain of the target sequence. Again, the second target domaincomprises n nucleotides, wherein n is an integer of at least 1, andpreferably from 1 to 100 s, with from 1 to 10 being preferred and from1, 2, 3, 4 and 5 being particularly preferred. What is important is thatthe first and third target domains are non-contiguous, e.g. notadjacent.

In a preferred embodiment, the first probe, further comprises, 3′ to thesecond domain, an interrogation position within the 3′ six terminalbases. As used herein, the base which basepairs with a detectionposition base in a hybrid is termed a “readout position” or an“interrogation position”; thus one or the other of the first or secondprobes of the invention comprise an interrogation position, as outlinedherein. In some cases, when two SNP positions or detection positions arebeing elucidated, both the first and the second probes may compriseinterrogation positions.

When the first probe comprises the interrogation position, it fallswithin the six 3′ terminal nucleotides, with in three, and preferablytwo, and most preferably it is the 3′ terminal nucleotide. In somepreferred embodiments the interrogator is not the terminal position ofthe primer, but rather resides at a position 1, 2, 3, 4, 5 or 6nucleotides from the 3′ terminus of the primer. Alternatively, the firstprobe does not contain the interrogation position; rather the secondprobe does. This depends on whether the extension enzyme or the ligationenzyme is to confer the specificity required for the genotypingreaction.

In addition to the first probes of the invention, the compositions ofthe invention further comprise a second probe for each target sequence.The second probes each comprise a first domain comprising a sequencesubstantially complementary to the third target domain of a targetsequence as outlined herein.

In some embodiments, the second probes comprise a second universalpriming site. As outlined herein, the first and second probes cancomprise two universal primers, one in each orientation, for use in PCRreactions or other amplification reactions utilizing two primers. Thatis, as is known in the art, the orientation of primers is such to allowexponential amplification, such that the first universal primingsequence is in the “sense” orientation and the second universal primingsequence is in the “antisense” orientation.

In a preferred embodiment, it is the second probe that comprises theinterrogation position. In this embodiment, the second probe comprises a5′ interrogation nucleotide, although in some instances, depending onthe ligase, the interrogation nucleotide may be within 1-3 bases of the5′ terminus. However, it is preferred that the interrogation base be the5′ base.

In a preferred embodiment, either the first or second probe furthercomprises an adapter sequence, (sometimes referred to in the art as “zipcodes”) to allow the use of “universal arrays”. That is, arrays aregenerated that contain capture probes that are not target specific, butrather specific to individual artificial adapter sequences.

It should be noted that when two universal priming sequences and anadapter is used, the orientation of the construct should be such thatthe adapter gets amplified; that is, the two universal priming sequencesare generally at the termini of the amplification template, describedbelow.

The first and second probes are added to the target sequences to form afirst hybridization complexes. The first hybridization complexes arecontacted with a first universal primer that hybridizes to the firstuniversal priming sequence, an extension enzyme and dNTPs.

If it is the first probe that comprises the interrogation nucleotide, ofthe base at the interrogation position is perfectly complementary withthe base at the detection position, extension of the first primer occursthrough the second target domain, stopping at the 5′ of the secondprobe, to form extended first probes that are hybridized to the targetsequence, forming second hybridization complexes. If, however, the baseat the interrogation position is not perfectly complementary with thebase at the detection position, extension of the first probe will notoccur, and no subsequent amplification or detection will occur.

Extension of the enzyme will also occur if it is the second probe thatcomprises the interrogation position.

Once extended, the extended first probe is adjacent to the 5′ end of thesecond probe. In the case where the interrogation position was in thefirst probe, the two ends of the probes (the 3′ end of the first probeand the 5′ end of the second probe) are respectively perfectlycomplementary to the target sequence at these positions, and the twoprobes can be ligated together with a suitable ligase to formamplification templates.

The conditions for carrying out the ligation will depend on theparticular ligase used and will generally follow the manufacturer'srecommendations.

If, however, it is the second probe that carries the interrogationposition at its 5′ end, the base at the interrogation position must beperfectly complementary to the detection position in the target sequenceto allow ligation. In the absence of perfect complementarity, nosignificant ligation will occur between the extended first probe and thesecond probe.

It should be noted that the enzymes may be added sequentially orsimultaneously. If the target sequences are attached to a solid support,washing steps may also be incorporated if required.

The ligation of the extended first probe and the second probe results inan amplification template comprising at least one, and preferably two,universal primers and an optional adapter. Amplification can then bedone, in a wide variety of ways. As will be appreciated by those in theart, there are a wide variety of suitable amplification techniquesrequiring either one or two primers, as is generally outlined in U.S.Ser. No. 09/517,945, hereby expressly incorporated by reference.

Detection Systems

All of the methods and compositions herein are drawn to methods ofdetecting, quantifying and/or determining the base at the detectionposition of a target nucleic acid, generally by having differentialreactions occur depending on the presence or absence of a mismatch. Thereaction products are generally detected on arrays as is outlinedherein, although a number of different detection methods may be used.

As is more fully outlined below, preferred systems of the invention workas follows. An amplicon is attached (via hybridization) to an arraysite. This attachment is generally a direct hybridization between aadapter on the amplicon and a corresponding capture probe, although insome instances, the system can rely on indirect “sandwich” complexesusing capture extender probes as are known in the art. In a preferredembodiment, the target sequence (e.g. the amplicon) itself comprises thelabels. Alternatively, a label probe is added, that will hybridize to alabel sequence on the amplicon, forming an assay complex. The captureprobes of the array are substantially (and preferably perfectly)complementary to the adapter sequences.

The terms length determination, separation-by-length assay, andseparation-by-length assay medium are taken collectively to mean aprocess and its related apparatus that achieves separation of DNAfragments on the basis of length, size, mass, or any other physicalproperty. This includes generally, liquid chromatography,electrophoresis and direct mass spectrometry; more particularly, highperformance liquid chromatography (HPLC) and capillary electrophoresisor gel electrophoresis, and MALDI-TOF MS respectively.

Other detection assays or formats include classical configurations suchas the “dot-blot”. This method of hybridization gained wide-spread use,and many versions were developed (see M. L. M. Anderson and B. D. Young,in Nucleic Acid Hybridization-A Practical Approach, B. D. Hames and S.J. Higgins, Eds., IRL Press, Washington D.C., Chapter 4, pp. 73-111,1985). The “dot blot” hybridization has been further developed formultiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, inEPA 0228075, Jul. 8, 1987) and for the detection of overlapping clonesand the construction of genomic maps (G. A. Evans, in U.S. Pat. No.5,219,726, Jun. 15, 1993).

Another format, the so-called “sandwich” hybridization, involvesattaching oligonucleotide probes covalently to a solid support and usingthem to capture and detect multiple nucleic acid targets. (M. Ranki etal., Gene, 21, pp. 77-85, 1983; A. M. Palva, T. M. Ranki, and H. E.Soderlund, in UK Patent Application GB 2156074A, Oct. 2, 1985; T. M.Ranki and H. E. Soderlund in U.S. Pat. No. 4,563,419, Jan. 7, 1986; A.D. B. Malcolm and J. A. Langdale, in PCT WO 86/03782, Jul. 3, 1986; Y.Stabinsky, in U.S. Pat. No. 4,751,177, Jan. 14, 1988; T. H. Adams etal., in PCT WO 90/01564, Feb. 22, 1990; R. B. Wallace et al. 6 NucleicAcid Res. 11, p. 3543, 1979; and B. J. Connor et al., 80 Proc. Natl.Acad. Sci. USA pp. 278-282, 1983). Multiplex versions of these formatsare called “reverse dot blots”.

In another approach of matrix hybridization, Beattie et al., in The 1992San Diego Conference: Genetic Recognition, November, 1992, used amicrorobotic system to deposit micro-droplets containing specific DNAsequences into individual microfabricated sample wells on a glasssubstrate. The hybridization in each sample well is detected byinterrogating miniature electrode test fixtures, which surround eachindividual microwell with an alternating current (AC) electric field.

One preferred aspect of the present invention is that it results inhigh-throughput screening capabilities. In the assays described herein,from a few up to millions of different tags identifying, e.g., SNPs, canbe identified simultaneously. For example, using simple dot-blothybridization methods, membranes with thousands of immobilized probescan be generated for screening against tags. The solid-phase techniquesdescribed below can be adapted to having literally millions of differentimmobilized nucleic acids per square inch. Similarly, very large sets ofamplified DNAs, e.g., tags, can be immobilized on membranes forsimultaneous screening against one or more sequence.

In one embodiment, the identity of the amplification products aredetermined by detecting the molecular weights of the amplificationproduct or a fragment thereof, such as by chromatography or massspectroscopy.

For instance, the gross molecular weight of an amplification product ora discrete fragment thereof can be detected. As set forth above, eachmember of a probe library (i.e., all of the probes in the reaction) hasa unique molecular weight label based on the particular sequence of thetag. For instance, mass spectrometry can provide high detectionsensitivity and accuracy of mass measurements that can discern betweenprobes which, while identical in length, differ in sequence by onlybase. Thus, complex libraries can be constructed by calculating theoverall molecular weight of each amplification product to be detected byvarying the G/C/A/T content in the tag sequence. In certain preferredembodiments, the nucleic acid sequence which is being detected includes,as its only variable sequence, the tag sequence and not the templatehomology regions. Such fragments can be generated, for example, byincluding restriction sites that flank the tag sequence, or choosing thePCR primers such that only the tag sequence is the only variable regionof the covalently closed circular product which is included in theamplification products. That being said, in those embodiments where theamplification product which is being detected also includes the templatehomology region(s), the calculation and design of the tag sequences willneed to include the variability in the THRs as well in order to produceproducts having a unique molecular weight so as to be discernable fromone another by mass spectroscopy or other detection means as may bechosen.

Those skilled in the art will recognize that very simple algorithms canbe used to calculate the molecular weights for each member of a libraryby varying the sequence of the tag, taking into account if necessary thesequences of the template homology regions. The molecular weightcomplexity of the tag can be increased by allowing the probes to vary inlength as well sequence.

In certain instances, the library can be deconvoluted by chromatographictechniques prior to detection by mass spectroscopy. For example, priorto introducing a sample into the spectrometer, the mixture can first beat least semi-purified. Separation procedures based on size (e.g.gel-filtration), solubility (e.g. isoelectric precipitation) or electriccharge (e.g. electrophoresis, isoelectric focusing, ion exchangechromatography) may be used to separate a mixture of amplimers. Apreferred separation procedure is high performance liquid chromatography(HPLC).

In certain embodiments, the amplification product can include anintegrated mass label for multiplex sequencing. Multiplexing by massmodification in this case is obtained by mass-modifying the nucleic acidprimer, e.g., at the level of the sugar or base moiety. Such embodimentsare most practical when amplification products are to be mixed fordetection after the amplification step rather than before.

Suitable mass spectrometry techniques for use in the present inventioninclude DNA analyses of the present invention include collision-induceddissociation (CID) fragmentation analysis (e.g., CID in conjunction witha MS/MS configuration, see Schram, K. (1990) “Mass Spectrometry ofNucleic Acid Components,” in Biomedical Applications of MassSpectrometry 34:203-287; and Crain P. (1990) Mass Spectrometry Reviews9:505-554); fast atomic bombardment (FAB mass spectrometry) and plasmadesorption (PD mass spectrometry), see Koster et al. (1987) BiomedicalEnvironmental Mass Spectrometry 14:111-116; and electrospray/ionspray(ES) and matrix-assisted laser desorption/ionization (MALDI) massspectrometry (see Fenn et al. (1984) J. Phys. Chem. 88:4451-4459, Smithet al. (1990) Anal. Chem. 62:882-889, and Ardrey, B. (1992) SpectroscopyEurope 4:10-18). MALDI mass spectrometry is particularly well suited tosuch analyses when a time-of-flight (TOF) configuration is used as amass analyzer (MALDI-TOF). See International Publication No. WO97/33000, published Sep. 12, 1997, see also Huth-Fehre et al. (1992)Rapid Communications in Mass Spectrometry 6:209-213, and Williams et al.(1990) Rapid Communications in Mass Spectrometry 4:348-351.

Suitable mass spectrometry techniques for use in the mass tag analysesof the present invention include collision-induced dissociation (CID)fragmentation analysis (e.g., CID in conjunction with a MS/MSconfiguration, see Schram, K. (1990) “Mass Spectrometry of Nucleic AcidComponents,” in Biomedical Applications of Mass Spectrometry 34:203-287;and Crain P. (1990) Mass Spectrometry Reviews 9:505-554); fast atomicbombardment (FAB mass spectrometry) and plasma desorption (PD massspectrometry), see Koster et al. (1987 Biomedical Environmental MassSpectrometry 14:111-116; and electrospray/ionspray (ES) andmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(see Fenn et al. (1984) J. Phys. Chem. 88:4451-4459, Smith et al. (1990)Anal. Chem. 62:882-889, and Ardrey, B. (1992) Spectroscopy Europe4:10-18). MALDI mass spectrometry is particularly well suited to suchanalyses when a time-of-flight (TOF) configuration is used as a massanalyzer (MALDI-TOF). See International Publication No. WO 97/33000,published Sep. 12, 1997, see also Huth-Fehre et al. (1992) RapidCommunications in Mass Spectrometry 6:209-213, and Williams et al.(1990) Rapid Communications in Mass Spectrometry 4:348-351.

In this regard, a number of mass tags suitable for use with nucleicacids are known (see U.S. Pat. No. 5,003,059 to Brennan and U.S. Pat.No. 5,547,835 to Koster), including mass tags which are cleavable fromthe nucleic acid (see International Publication No. WO 97/27331).

In another embodiment, the hybridization tags are detected on amicro-formatted multiplex or matrix devices (e.g., DNA chips) (see M.Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp.757-758, 1992). These methods usually attach specific DNA sequences tovery small specific areas of a solid support, such as micro-wells of aDNA chip. In one variant, the invention is adapted to solid phase arraysfor the rapid and specific detection of multiple polymorphicnucleotides, e.g., SNPs. Typically, an oligonucleotide is linked to asolid support and a tag nucleic acid is hybridized to theoligonucleotide. Either the oligonucleotide, or the tag, or both, can belabeled, typically with a fluorophore. Where the tag is labeled,hybridization is detected by detecting bound fluorescence. Where theoligonucleotide is labeled, hybridization is typically detected byquenching of the label. Where both the oligonucleotide and the tag arelabeled, detection of hybridization is typically performed by monitoringa color shift resulting from proximity of the two bound labels. Avariety of labeling strategies, labels, and the like, particularly forfluorescent based applications are described, supra.

In one embodiment, an array of oligonucleotides are synthesized on asolid support. Exemplary solid supports include glass, plastics,polymers, metals, metalloids, ceramics, organics, etc. Using chipmasking technologies and photoprotective chemistry it is possible togenerate ordered arrays of nucleic acid probes. These arrays, which areknown, e.g., as “DNA chips,” or as very large scale immobilized polymerarrays (“VLSIPS™” arrays) can include millions of defined probe regionson a substrate having an area of about 1 cm2 to several cm2, therebyincorporating sets of from a few to millions of probes.

The construction and use of solid phase nucleic acid arrays to detecttarget nucleic acids is well described in the literature. See, Fodor etal. (1991) Science, 251: 767-777; Sheldon et al. (1993) ClinicalChemistry 39(4): 718-719; Kozal et al. (1996) Nature Medicine 2(7):753-759 and Hubbell U.S. Pat. No. 5,571,639. See also, Pinkel et al.PCT/US95/16155 (WO 96/17958). In brief, a combinatorial strategy allowsfor the synthesis of arrays containing a large number of probes using aminimal number of synthetic steps. For instance, it is possible tosynthesize and attach all possible DNA 8 mer oligonucleotides (48, or65,536 possible combinations) using only 32 chemical synthetic steps. Ingeneral, VLSIPS™ procedures provide a method of producing 4n differentoligonucleotide probes on an array using only 4n synthetic steps.

Light-directed combinatorial synthesis of oligonucleotide arrays on aglass surface is performed with automated phosphoramidite chemistry andchip masking techniques similar to photoresist technologies in thecomputer chip industry. Typically, a glass surface is derivatized with asilane reagent containing a functional group, e.g., a hydroxyl or aminegroup blocked by a photolabile protecting group. Photolysis through aphotolithogaphic mask is used selectively to expose functional groupswhich are then ready to react with incoming 5′-photoprotected nucleosidephosphoramidites. The phosphoramidites react only with those sites whichare illuminated (and thus exposed by removal of the photolabile blockinggroup). Thus, the phosphoramidites only add to those areas selectivelyexposed from the preceding step. These steps are repeated until thedesired array of sequences have been synthesized on the solid surface.

A 96 well automated multiplex oligonucleotide synthesizer (A.M.O.S.) hasalso been developed and is capable of making thousands ofoligonucleotides (Lashkari et al. (1995) PNAS 93: 7912). Existinglight-directed synthesis technology can generate high-density arrayscontaining over 65,000 oligonucleotides (Lipshutz et al. (1995) BioTech.19: 442.

Combinatorial synthesis of different oligonucleotide analogues atdifferent locations on the array is determined by the pattern ofillumination during synthesis and the order of addition of couplingreagents. Monitoring of hybridization of target nucleic acids to thearray is typically performed with fluorescence microscopes or laserscanning microscopes. In addition to being able to design, build and useprobe arrays using available techniques, one of skill is also able toorder custom-made arrays and array-reading devices from manufacturersspecializing in array manufacture. For example, Affymetrix Corp., inSanta Clara, Calif. manufactures DNA VLSIP™ arrays.

It will be appreciated that oligonucleotide design is influenced by theintended application. For example, where several oligonucleotide-taginteractions are to be detected in a single assay, e.g., on a single DNAchip, it is desirable to have similar melting temperatures for all ofthe probes. Accordingly, the length of the probes are adjusted so thatthe melting temperatures for all of the probes on the array are closelysimilar (it will be appreciated that different lengths for differentprobes may be needed to achieve a particular T[m] where different probeshave different GC contents). Although melting temperature is a primaryconsideration in probe design, other factors are optionally used tofurther adjust probe construction, such as selecting against primerself-complementarity and the like. The “active” nature of the devicesprovide independent electronic control over all aspects of thehybridization reaction (or any other affinity reaction) occurring ateach specific microlocation. These devices provide a new mechanism foraffecting hybridization reactions which is called electronic stringencycontrol (ESC). For DNA hybridization reactions which require differentstringency conditions, ESC overcomes the inherent limitation ofconventional array technologies. The active devices of this inventioncan electronically produce “different stringency conditions” at eachmicrolocation. Thus, all hybridizations can be carried out optimally inthe same bulk solution. These arrays are described in U.S. Pat. No.6,051,380 by Sosnowski et al.

Accordingly, the present invention provides array compositionscomprising at least a first substrate with a surface comprisingindividual sites. By “array” or “biochip” herein is meant a plurality ofnucleic acids in an array format; the size of the array will depend onthe composition and end use of the array. Nucleic acids arrays are knownin the art, and can be classified in a number of ways; both orderedarrays (e.g. the ability to resolve chemistries at discrete sites), andrandom arrays (e.g. bead arrays) are included. Ordered arrays include,but are not limited to, those made using photolithography techniques(Affymetrix GeneChip™), spotting techniques (Synteni and others),printing techniques (Hewlett Packard and Rosetta), electrode arrays,three dimensional “gel pad” arrays, etc. Liquid arrays may also be used,i.e. three-dimensional array methods such as flow cytometry. When flowcytometry is the detection method, amplicons are immobilized to asupport such as a microsphere as described herein. The microspheres areapplied to a flow cytometer and the amplicons are detected optically asdescribed herein.

In a preferred embodiment, when beads are used, the beads aredistributed in or on an additional support or substrate is generallyflat (planar), although as will be appreciated by those in the art,other configurations of substrates may be used as well; for example,three dimensional configurations can be used, for example by embeddingthe beads in a porous block of plastic that allows sample access to thebeads and using a confocal microscope for detection. Similarly, thebeads may be placed on the inside surface of a tube, for flow-throughsample analysis to minimize sample volume. Preferred substrates includeoptical fiber bundles as discussed below, and flat planar substratessuch as glass, polystyrene and other plastics and acrylics. In apreferred embodiment such substrates include multi-well plates as areknown in the art. In a preferred embodiment magnetic force is used toimmobilized magnetic beads on the solid support.

A preferred embodiment utilizes microspheres on a variety of arraysubstrates including fiber optic bundles, as are outlined in PCTsUS98/21193, PCT US99/14387 and PCT US98/05025; WO98/50782; and U.S. Ser.Nos. 09/287,573, 09/151,877, 09/256,943, 09/316,154, 60/119,323,09/315,584; all of which are expressly incorporated by reference. Whilemuch of the discussion below is directed to the use of microspherearrays on fiber optic bundles, any array format of nucleic acids onsolid supports may be utilized.

Arrays containing from about 2 different bioactive agents (e.g.different beads, when beads are used) to many millions can be made, withvery large arrays being possible. Generally, the array will comprisefrom two to as many as a billion or more, depending on the size of thebeads and the array substrate, as well as the end use of the array, thusvery high density, high density, moderate density, low density and verylow density arrays may be made. Preferred ranges for very high densityarrays are from about 10,000,000 to about 2,000,000,000, with from about100,000,000 to about 1,000,000,000 being preferred (all numbers being insquare cm). High density arrays range about 100,000 to about 10,000,000,with from about 1,000,000 to about 5,000,000 being particularlypreferred. Moderate density arrays range from about 10,000 to about100,000 being particularly preferred, and from about 20,000 to about50,000 being especially preferred. Low density arrays are generally lessthan 10,000, with from about 1,000 to about 5,000 being preferred. Verylow density arrays are less than 1,000, with from about 10 to about 1000being preferred, and from about 100 to about 500 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single bioactive agent may be made as well. In addition, insome arrays, multiple array substrates may be used, either of differentor identical compositions. Thus for example, large arrays may comprise aplurality of smaller array substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 μm orless (with beads of 200 nm possible) can be used, and very small fibersare known, it is possible to have as many as 40,000 or more (in someinstances, 1 million) different elements (e.g. fibers and beads) in a 1mm² fiber optic bundle, with densities of greater than 25,000,000individual beads and fibers (again, in some instances as many as 50-100million) per 0.5 cm obtainable (4 million per square cm for 5μcenter-to-center and 100 million per square cm for 1μ center-to-center).

By “array substrate” or “array solid support” or other grammaticalequivalents herein is meant any material that can be modified to containdiscrete individual sites appropriate for the attachment or associationof beads and is amenable to at least one detection method. As will beappreciated by those in the art, the number of possible array substratesis very large. Possible array substrates include, but are not limitedto, glass and modified or functionalized glass, plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, etc.),polysaccharides, nylon or nitrocellulose, resins, silica or silica-basedmaterials including silicon and modified silicon, carbon, metals,inorganic glasses, plastics, optical fiber bundles, and a variety ofother polymers. In general, the array substrates allow optical detectionand do not themselves appreciably fluoresce.

Generally the array substrate is flat (planar), although as will beappreciated by those in the art, other configurations of arraysubstrates may be used as well; for example, three dimensionalconfigurations can be used, for example by embedding the beads in aporous block of plastic that allows sample access to the beads and usinga confocal microscope for detection. Similarly, the beads may be placedon the inside surface of a tube, for flow-through sample analysis tominimize sample volume. Preferred array substrates include optical fiberbundles as discussed below, and flat planar array substrates such aspaper, glass, polystyrene and other plastics and acrylics.

In a preferred embodiment, the array substrate is an optical fiberbundle or array, as is generally described in U.S. Ser. Nos. 08/944,850and 08/519,062, PCT US98/05025, and PCT US98/09163, all of which areexpressly incorporated herein by reference. Preferred embodimentsutilize preformed unitary fiber optic arrays. By “preformed unitaryfiber optic array” herein is meant an array of discrete individual fiberoptic strands that are co-axially disposed and joined along theirlengths. The fiber strands are generally individually clad. However, onething that distinguished a preformed unitary array from other fiberoptic formats is that the fibers are not individually physicallymanipulatable; that is, one strand generally cannot be physicallyseparated at any point along its length from another fiber strand.

Generally, the arrayed array compositions of the invention can beconfigured in several ways; see for example U.S. Ser. No. 09/473,904,hereby expressly incorporated by reference. In a preferred embodiment,as is more fully outlined below, a “one component” system is used. Thatis, a first array substrate comprising a plurality of assay locations(sometimes also referred to herein as “assay wells”), such as amicrotiter plate, is configured such that each assay location containsan individual array. That is, the assay location and the array locationare the same. For example, the plastic material of the microtiter platecan be formed to contain a plurality of “bead wells” in the bottom ofeach of the assay wells. Beads containing the capture probes of theinvention can then be loaded into the bead wells in each assay locationas is more fully described below.

Alternatively, a “two component” system can be used. In this embodiment,the individual arrays are formed on a second array substrate, which thencan be fitted or “dipped” into the first microtiter plate substrate. Apreferred embodiment utilizes fiber optic bundles as the individualarrays, generally with “bead wells” etched into one surface of eachindividual fiber, such that the beads containing the capture probes areloaded onto the end of the fiber optic bundle. The composite array thuscomprises a number of individual arrays that are configured to fitwithin the wells of a microtiter plate.

By “composite array” or “combination array” or grammatical equivalentsherein is meant a plurality of individual arrays, as outlined above.Generally the number of individual arrays is set by the size of themicrotiter plate used; thus, 96 well, 384 well and 1536 well microtiterplates utilize composite arrays comprising 96, 384 and 1536 individualarrays, although as will be appreciated by those in the art, not eachmicrotiter well need contain an individual array. It should be notedthat the composite arrays can comprise individual arrays that areidentical, similar or different. That is, in some embodiments, it may bedesirable to do the same 2,000 assays on 96 different samples;alternatively, doing 192,000 experiments on the same sample (i.e. thesame sample in each of the 96 wells) may be desirable. Alternatively,each row or column of the composite array could be the same, forredundancy/quality control. As will be appreciated by those in the art,there are a variety of ways to configure the system. In addition, therandom nature of the arrays may mean that the same population of beadsmay be added to two different array surfaces, resulting in substantiallysimilar but perhaps not identical arrays.

At least one surface of the array substrate is modified to containdiscrete, individual sites for later association of microspheres. Thesesites may comprise physically altered sites, i.e. physicalconfigurations such as wells or small depressions in the array substratethat can retain the beads, such that a microsphere can rest in the well,or the use of other forces (magnetic or compressive), or chemicallyaltered or active sites, such as chemically functionalized sites,electrostatically altered sites, hydrophobically/hydrophilicallyfunctionalized sites, spots of adhesive, etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the arraysubstrate. However, it should be noted that these sites may not bediscrete sites. That is, it is possible to use a uniform surface ofadhesive or chemical functionalities, for example, that allows theattachment of beads at any position. That is, the surface of the arraysubstrate is modified to allow attachment of the microspheres atindividual sites, whether or not those sites are contiguous ornon-contiguous with other sites. Thus, the surface of the arraysubstrate may be modified such that discrete sites are formed that canonly have a single associated bead, or alternatively, the surface of thearray substrate is modified and beads may go down anywhere, but they endup at discrete sites. That is, while beads need not occupy each site onthe array, no more than one bead occupies each site.

In a preferred embodiment, the surface of the array substrate ismodified to contain wells, i.e. depressions in the surface of the arraysubstrate. This may be done as is generally known in the art using avariety of techniques, including, but not limited to, photolithography,stamping techniques, molding techniques and microetching techniques. Aswill be appreciated by those in the art, the technique used will dependon the composition and shape of the array substrate.

In a preferred embodiment, physical alterations are made in a surface ofthe array substrate to produce the sites. In a preferred embodiment, thearray substrate is a fiber optic bundle and the surface of the arraysubstrate is a terminal end of the fiber bundle, as is generallydescribed in 08/818,199 and 09/151,877, both of which are herebyexpressly incorporated by reference. In this embodiment, wells are madein a terminal or distal end of a fiber optic bundle comprisingindividual fibers. In this embodiment, the cores of the individualfibers are etched, with respect to the cladding, such that small wellsor depressions are formed at one end of the fibers. The required depthof the wells will depend on the size of the beads to be added to thewells.

Generally in this embodiment, the microspheres are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

In a preferred embodiment, the surface of the array substrate ismodified to contain chemically modified sites, that can be used toattach, either covalently or non-covalently, the microspheres of theinvention to the discrete sites or locations on the array substrate.“Chemically modified sites” in this context includes, but is not limitedto, the addition of a pattern of chemical functional groups includingamino groups, carboxy groups, oxo groups and thiol groups, that can beused to covalently attach microspheres, which generally also containcorresponding reactive functional groups; the addition of a pattern ofadhesive that can be used to bind the microspheres (either by priorchemical functionalization for the addition of the adhesive or directaddition of the adhesive); the addition of a pattern of charged groups(similar to the chemical functionalities) for the electrostaticattachment of the microspheres, i.e. when the microspheres comprisecharged groups opposite to the sites; the addition of a pattern ofchemical functional groups that renders the sites differentiallyhydrophobic or hydrophilic, such that the addition of similarlyhydrophobic or hydrophilic microspheres under suitable experimentalconditions will result in association of the microspheres to the siteson the basis of hydroaffinity. For example, the use of hydrophobic siteswith hydrophobic beads, in an aqueous system, drives the association ofthe beads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow attachment of the beads at discrete sites, as well as treatment ofthe surface resulting in discrete sites. As will be appreciated by thosein the art, this may be accomplished in a variety of ways.

In some embodiments, the beads are not associated with an arraysubstrate. That is, the beads are in solution or are not distributed ona patterned substrate.

In a preferred embodiment, the compositions of the invention furthercomprise a population of microspheres. By “population” herein is meant aplurality of beads as outlined above for arrays. Within the populationare separate subpopulations, which can be a single microsphere ormultiple identical microspheres. That is, in some embodiments, as ismore fully outlined below, the array may contain only a single bead foreach capture probe; preferred embodiments utilize a plurality of beadsof each type.

By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on the class of capture probe and the method ofsynthesis. Suitable bead compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphite, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and Teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers Ind. is a helpfulguide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either capture probe attachment or tagattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

It should be noted that a key component of the invention is the use ofan array substrate/bead pairing that allows the association orattachment of the beads at discrete sites on the surface of the arraysubstrate, such that the beads do not move during the course of theassay.

Each microsphere comprises a capture probe, although as will beappreciated by those in the art, there may be some microspheres which donot contain a capture probe, depending on the synthetic methods.

Attachment of the nucleic acids may be done in a variety of ways, aswill be appreciated by those in the art, including, but not limited to,chemical or affinity capture (for example, including the incorporationof derivatized nucleotides such as AminoLink or biotinylated nucleotidesthat can then be used to attach the nucleic acid to a surface, as wellas affinity capture by hybridization), cross-linking, and electrostaticattachment, etc. In a preferred embodiment, affinity capture is used toattach the nucleic acids to the beads. For example, nucleic acids can bederivatized, for example with one member of a binding pair, and thebeads derivatized with the other member of a binding pair. Suitablebinding pairs are as described herein for IBL/DBL pairs. For example,the nucleic acids may be biotinylated (for example using enzymaticincorporate of biotinylated nucleotides, for by photoactivatedcross-linking of biotin). Biotinylated nucleic acids can then becaptured on streptavidin-coated beads, as is known in the art.Similarly, other hapten-receptor combinations can be used, such asdigoxigenin and anti-digoxigenin antibodies. Alternatively, chemicalgroups can be added in the form of derivatized nucleotides, that canthem be used to add the nucleic acid to the surface.

Preferred attachments are covalent, although even relatively weakinteractions (i.e. non-covalent) can be sufficient to attach a nucleicacid to a surface, if there are multiple sites of attachment per eachnucleic acid. Thus, for example, electrostatic interactions can be usedfor attachment, for example by having beads carrying the opposite chargeto the bioactive agent.

Similarly, affinity capture utilizing hybridization can be used toattach nucleic acids to beads.

Alternatively, chemical crosslinking may be done, for example byphotoactivated crosslinking of thymidine to reactive groups, as is knownin the art.

In a preferred embodiment, each bead comprises a single type of captureprobe, although a plurality of individual capture probes are preferablyattached to each bead. Similarly, preferred embodiments utilize morethan one microsphere containing a unique capture probe; that is, thereis redundancy built into the system by the use of subpopulations ofmicrospheres, each microsphere in the subpopulation containing the samecapture probe.

As will be appreciated by those in the art, the capture probes mayeither be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the capture probes to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

In a preferred embodiment, the capture probes are synthesized directlyon the beads. As is known in the art, many classes of chemical compoundsare currently synthesized on solid supports, such as peptides, organicmoieties, and nucleic acids. It is a relatively straightforward matterto adjust the current synthetic techniques to use beads.

In a preferred embodiment, the capture probes are synthesized first, andthen covalently attached to the beads. As will be appreciated by thosein the art, this will be done depending on the composition of thecapture probes and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres include, but are not limited to, amino groups includingaliphatic and aromatic amines, carboxylic acids, aldehydes, amides,chloromethyl groups, hydrazide, hydroxyl groups, sulfonates andsulfates.

In general, the methods of making the arrays and of decoding the arraysis done to maximize the number of different candidate agents that can beuniquely encoded. The compositions of the invention may be made in avariety of ways. In general, the arrays are made by adding a solution orslurry comprising the beads to a surface containing the sites forattachment of the beads. This may be done in a variety of buffers,including aqueous and organic solvents, and mixtures. The solvent canevaporate, and excess beads are removed.

In a preferred embodiment, when non-covalent methods are used toassociate the beads with the array, a novel method of loading the beadsonto the array is used. This method comprises exposing the array to asolution of particles (including microspheres and cells) and thenapplying energy, e.g. agitating or vibrating the mixture. In a preferredembodiment when the array substrate is a fiber optic bundle, the arraysubstrate is tapped into the beads. That is, the energy is tapping. Thisresults in an array comprising more tightly associated particles, as theagitation is done with sufficient energy to cause weakly-associatedbeads to fall off (or out, in the case of wells). These sites are thenavailable to bind a different bead. In this way, beads that exhibit ahigh affinity for the sites are selected. Arrays made in this way havetwo main advantages as compared to a more static loading: first of all,a higher percentage of the sites can be filled easily, and secondly, thearrays thus loaded show a substantial decrease in bead loss duringassays. Thus, in a preferred embodiment, these methods are used togenerate arrays that have at least about 50% of the sites filled, withat least about 75% being preferred, and at least about 90% beingparticularly preferred. Similarly, arrays generated in this mannerpreferably lose less than about 20% of the beads during an assay, withless than about 10% being preferred and less than about 5% beingparticularly preferred.

Methods of adding, washing and detecting the amplicons on the array arewell known.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the present invention finds use in thequantification of PCR reactions. Thus, the invention provides a methodfor quantifying the number of one or more specific sequences in a sampleof nucleic acids. The method may be similar to any of the methodsdescribed above, so long as the product being detected is present inproportions that are directly correlated with the amount of originaltemplate sequence. This is the case, e.g., where the method involves ahybridization step to the template DNA, circularization of the probe,extension of the primers and detection of the extension product. In apreferred embodiment, the method further comprises an amplificationstep, wherein the amplification reaction is a controlled amplification.This is the case, e.g., when using PCR amplification and stopping thePCR reaction during the exponential phase. The amount of amplifiedproduct in this situation will be directly proportional to the amount oforiginal sequence in the nucleic acid sample. Thus, in a preferredembodiment, several amplification reactions are conducted in parallel,using a different number of amplification cycles in each of them. Thiswill assure that at least one of the reactions will have been stopped inthe exponential phase.

In methods for quantifying the number of a specific sequence in asample, it may also be desirable in certain situations to include amarker nucleic acid. The marker nucleic acid can be added to thereaction during the hybridization stage or at any stage thereafter andbe subject or not to the same reactions. Alternatively, the marker DNAis used merely to determine the amount of applied product at the end ofthe amplification step.

The methods for genotyping and those for quantifying can be usedsimultaneously, so long as the processes are controlled, such that theamount of amplified product is directly correlated to the amount of theoriginal sequence in the sample nucleic acid.

All references cited herein are expressly incorporated by reference.

1-14. (canceled)
 15. A method for detecting different target nucleicacid sequences of interest in a sample, each sequence comprising from 3′to 5′, contiguous first, second, and third target domains, wherein thefirst target domain has a detection position one, two, three or fournucleotides from the 3′ terminal base of the second target domain, andthe second target domain is at least one nucleotide, comprising thesteps of: (a) providing a sample having different target nucleic acidsequences of interest; (b) contacting the sample with dNTPs and primersthat hybridize to the first target domains, and extending the primers toobtain first extension products; (c) contacting the first extensionproducts with a set of probes for each of the different target nucleicacid sequences of interest to form hybridization complexes, each setcomprising: a first probe comprising from 5′ to 3′: a first universalpriming sequence and a sequence that is substantially complementary tothe first target domain and that has an interrogation position suitablefor basepairing with the detection position; and a second probecomprising 5′ to 3′: a sequence substantially complementary to the thirdtarget domain, and a second universal priming sequence, wherein at leastone probe contains a distinct adapter sequence not native to the targetsequence of interest; (d) contacting the hybridization complexes with anextension enzyme and dNTPs, wherein for each hybridization complex, ifthe base at the interrogation position is perfectly complementary to thebase at the detection position, then the first probe is extended alongthe second target domain; (e) ligating the extended first probes tosecond probes to form amplification templates; (f) amplifying theamplification templates with first and second universal primers toproduce amplicons; (g) immobilizing the amplicons on solid phase captureprobes that are specific to individual adapter sequences; and (h)detecting the presence of different immobilized amplicons at the captureprobes; thereby indicating the presence of the different targetsequences of interest in the sample.
 16. The method of claim 15, whereinthe first extension product is purified from the sample.
 17. The methodof claim 16, wherein the dNTPs are labeled, and the first extensionproduct is purified by means of the label.
 18. The method of claim 16,wherein the primer is tagged with a label, and the first extensionproduct is purified by means of the label.
 19. The method of claim 18,wherein the primer is tagged with biotin or streptavidin.
 20. The methodof claim 18, wherein prior to step (c), the first extension products areimmobilized to a solid support.
 21. The method of claim 15, wherein thenucleic acids in step (a) comprise single nucleotide polymorphismalleles.
 22. The method of claim 15, wherein said target sequencescomprise single nucleotide polymorphism alleles and said probe setscomprise allele-specific probes that discriminate between said alleles.23. The method of claim 15, wherein a set of probes for each of at least100 different target sequences of interest are contacted in step (b),thereby indicating of the presence of the at least 100 different targetsequences of interest in the sample.
 24. The method of claim 15, whereina set of probes for each of at least 200 different target sequences ofinterest are contacted in step (b), thereby indicating of the presenceof the at least 200 different target sequences of interest in thesample.
 25. The method of claim 15, wherein the detection position isone nucleotide from the 3′-terminal base of the second domain.
 26. Themethod of claim 15, wherein the detection position is two nucleotidesfrom the 3′-terminal base of the second domain.
 27. The method of claim15, wherein the second target domain is 1 nucleotide in length.
 28. Themethod of claim 15, wherein the second target domain is from 2 to 5nucleotides in length.
 29. The method of claim 15, wherein each set ofprobes further comprises a third probe comprising from 5′ to 3′: thethird universal priming sequence and a sequence that is substantiallycomplementary to the first target domain and that has an interrogationposition corresponding to the detection position, wherein theinterrogation position is not complementary to the base at the detectionposition, whereby the third probe is not extended in step (c).
 30. Themethod of claim 15, wherein each of said probe sets comprises auniversal priming sequence that is the same for other probe sets. 31.The method of claim 15, wherein a universal primer is detectablylabeled.
 32. The method of claim 15, wherein the first universal primersin step (e) comprise a detectable label.
 33. The method of claim 15,wherein the first probe comprises the adapter sequence, whereby anamplicon comprises from 5′ to 3′: the first universal primer, theadapter sequence, the first target domain, the second target domain, thethird target domain, and the second universal primer.
 34. The method ofclaim 15, wherein the second probe comprises the adapter sequence,whereby an amplicon comprises from 5′ to 3′: the first universal primer,the first target domain, the second target domain, the third targetdomain, the adapter sequence, and the second universal primer.
 35. Themethod of claim 15, wherein step (e) comprises performing PCR using asingle pair of universal primers that are complementary to said firstuniversal priming sequence and said second universal priming sequence.36. The method of claim 15, wherein the hybridization complexes arewashed while immobilized on the solid support under conditionssufficient to remove non-hybridized nucleic acids.
 37. The method ofclaim 15, wherein the amplification templates are immobilized on thesolid support and are washed under conditions sufficient to removenon-hybridized nucleic acids.
 38. The method of claim 15, wherein thesecond probe comprises a sequence that is complementary to the thirdtarget domain.
 39. The method of claim 38, wherein ligation in step (d)occurs only when the 3′-end of the extended first probe and the 5′-endof the second probe are perfectly complementary to the target sequence.40. The method of claim 15, wherein the second probe comprises aninterrogation position.
 41. The method of claim 40, wherein theinterrogation position is at the 5′ end of the second probe.
 42. Themethod of claim 15, wherein no significant ligation occurs between theextended first probe and the second probe unless the interrogationposition of the second probe is perfectly complementary to the targetsequence.
 43. The method of claim 15, wherein step (a) comprisesproviding double-stranded sample and denaturing the sample into separatestrands, and wherein the primers in step (b) comprise primers thathybridize to one strand and primers that hybridize to the other strand.